Methods for treating nervous system disorders

Abstract

Provided herein are methods for the treatment of a nervous system disorder, including epilepsy, using modified oligonucleotides that inhibit AMPAR.

Description

Attorney Docket No.01138-0045-00PCT METHODS FOR TREATING NERVOUS SYSTEM DISORDERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 453,814, filed March 22, 2023, which is incorporated by reference herein in its entirety for any purpose. FIELD

[0002] Provided herein methods for treating nervous system disorders, including epilepsy.   BACKGROUND

[0003] Epilepsy is a common neurological disorder that affects around 50 million people worldwide and about 0.5 to 1% of the general population long-term. Further, approximately 5% of the population have experienced at least one epileptic seizure at some point in their lives. Wahab, A., “Difficulties in Treatment and Management of Epilepsy and Challenges in New Drug Development,” Pharmaceuticals, Vol.3(7), July 2010.

[0004] Epilepsy is a serious disease, with mortality in epileptic patients being two- to three- times that of the general population. Epileptic patients also may suffer from various social, emotional, psychological, and medical challenges including social isolation, unemployment, psychological issues, reduced quality of life, and medical comorbidities due to medication and seizures.

[0005] There is presently no known cure for epilepsy; treatment is addressed to providing symptomatic relief and averting seizure episodes. For most patients, antiepileptic drugs are prescribed as a life-long treatment; however, long-term use of current therapies is limited by their adverse effects, withdrawal symptoms, interactions with other drugs, and economic burdens.

[0006] Additionally, it is reported that about 30% of all patients with epilepsy are resistant to pharmaceutical drug therapies, and about 15-35% of all patients with epilepsy will fail to achieve long-term remission. See Wahab.

[0007] While epileptic disorders have been treated for years and significant funding and research has been devoted to developing improved, antiepileptic drugs for many decades, the cellular-based mechanism for the disease state still remains largely a mystery. Recently, evidence has emerged that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor plays a role in epilepsy and epileptogenesis. Hanada, T., “The AMPA 1   Attorney Docket No.01138-0045-00PCT Receptor as a Therapeutic Target in Epilepsy: Preclinical and Clinical Evidence,” Dovepress, Vol.7(39-50), Sept.18, 2014.

[0008] Accordingly, there remains a need for improved pharmaceutical agents to treat epilepsy and other nervous system disorders. SUMMARY

[0009] The present disclosure is directed to methods of treating a nervous system disorder in a subject, optionally, of treating epilepsy, comprising administering to the subject a therapeutically-effective amount of a compound comprising a modified oligonucleotide that inhibits AMPAR. Embodiment 1. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically-effective amount of a modified oligonucleotide consisting of 4-15 linked nucleosides, wherein the last four nucleosides at the 3’ end of the modified oligonucleotide have the nucleobase sequence UUUG, and wherein at least two of the last four nucleosides at the 3’ end of the modified oligonucleotide are bicyclic nucleosides. Embodiment 2. The method of embodiment 1, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleosides of the modified oligonucleotide are bicyclic nucleosides. Embodiment 3. The method of embodiment 1 or embodiment 2, wherein each bicyclic nucleoside is independently selected from S-cEt nucleoside, LNA nucleoside, and ENA nucleoside. Embodiment 4. The method of embodiment 3, wherein each bicyclic nucleoside is S-cEt nucleoside. Embodiment 5. The method of any one of the preceding embodiments, wherein at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. Embodiment 6. The method of embodiment 5, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. Embodiment 7. The method of embodiment 5 or embodiment 6, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 8. The method of any one of embodiments 1-3, wherein the modified oligonucleotide consists of 4-9, 4-10, 4-11, 4-12, 4-13, or 4-14 linked nucleosides. Embodiment 9. The method of any one of the preceding embodiments, wherein the modified oligonucleotide consists of 4 linked nucleosides. 2   Attorney Docket No.01138-0045-00PCT Embodiment 10. The method of embodiment 9, wherein the modified oligonucleotide is UFUMUSGSor USUSUSGS, wherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides. Embodiment 11. The method of any one of embodiments 1-8, wherein the modified oligonucleotide consists of 9 linked nucleosides. Embodiment 12. The method of any one of embodiments 1-8 and 11, wherein the modified oligonucleotide comprises the nucleobase sequence 5’-AGCACUUUG-3’, wherein each cytosine is independently selected from a non-methylated cytosine and a 5-methylcytosine Embodiment 13. The method of embodiment 11 or embodiment 12, wherein the modified oligonucleotide has the following nucleoside pattern in the 5’ to 3’ orientation: NSNSNMNFNFNFNMNSNSwherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides. Embodiment 14. The method of any one of the preceding embodiments, wherein the modified oligonucleotide is selected from the modified oligonucleotides in Table 1. Embodiment 15. The method of any one of the preceding embodiments, wherein a pharmaceutically acceptable salt of the modified oligonucleotide is administered to the subject. Embodiment 16. The method of embodiment 15, wherein the pharmaceutically acceptable salt is a sodium salt. Embodiment 17. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically effective amount of a modified oligonucleotide having the structure: 3   Attorney Docket No.01138-0045-00PCTthereof. Embodiment 18. The method of embodiment 17, comprising administering to the subject a pharmaceutically acceptable salt of the modified oligonucleotide. Embodiment 19. The method of embodiment 18, comprising administering to the subject a sodium salt of the modified oligonucleotide. Embodiment 20. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically-effective amount of a modified oligonucleotide having the structure: 4   Attorney Docket No.01138-0045-00PCT. Embodiment 21. The method of any one of the preceding embodiments, wherein the modified oligonucleotide is formulated in a pharmaceutical composition for administration to the subject, wherein the pharmaceutical composition comprises the modified oligonucleotide in an aqueous solution. Embodiment 22. The method of embodiment 21, wherein the aqueous solution is a saline solution. Embodiment 23. The method of any one of the preceding embodiments wherein the nervous system disorder is hearing loss, motor disease, amyotrophic lateral sclerosis (ALS), 5   Attorney Docket No.01138-0045-00PCT pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke, or glioblastoma. Embodiment 24. The method of any one of the preceding embodiments, wherein the nervous system disorder is epilepsy. Embodiment 25. The method of any one of the preceding embodiments, comprising administering to the subject at least one additional therapy. Embodiment 26. The method of embodiment 25, wherein the additional therapy is an anti- epileptic drug. BRIEF DESCRIPTION OF FIGURES

[0010] Figures 1A and 1B are graphs showing the time (in sec) observed between latency to seizure (clonic and tonic, respectively), in mice dosed with 60 mpk, 65 mpk, and 70 mpk of pentylenetetrazole (PTZ) in the dose pilot study of Example 3.

[0011] Figure 2 is a graph of Racine scores over time following administration of 60 mpk, 65 mpk, and 70 mpk of PTZ to mice in the dose pilot study of Example 3.

[0012] Figure 3 is a bar graph of survival data 30 min after administration of 60 mpk, 65 mpk, and 70 mpk of PTZ to mice in the dose pilot study of Example 3.

[0013] Figure 4 is a graph of latency to tonic seizure data obtained from the study of Example 4 comparing the CNS effects of RG4326, a negative control compound (NCC), and perampanel on PTZ induced seizures in mice.

[0014] Figure 5 is a graph of latency to cardiac arrest (e.g., death) data obtained from the study of Example 4 comparing the CNS effects of RG4326, NCC, and perampanel on PTZ induced seizures in mice.

[0015] Figure 6 is a graph of Racine scoring data in 5 min internals up to 30 min post- PTZ administration obtained from the study of Example 4, comparing the CNS effects of RG4326, NCC, and perampanel on PTZ-induced seizures in mice.

[0016] Figure 7 shows survival data up to 30 min post-PTZ administration in the study of Example 4, comparing the CNS effects of RG4326, NCC, and perampanel on PTZ-induced seizures in mice.

[0017] Figure 8 shows data from the 6-Hz induced seizure model of Example 6, reporting the percentage of animals in each group exhibiting forelimb clonus seizures post 6-Hz current administration. 6   Attorney Docket No.01138-0045-00PCT

[0018] Figure 9 shows data obtained from the 6-Hz induced seizure model of Example 6, reporting the percentage of animals in each group exhibiting Straub tail seizures post the 6-Hz current administration.

[0019] Figure 10 shows data obtained from the 6-Hz induced seizure model of Example 7 reporting the number of mice in each group exhibiting Straub tail seizures post the 6-Hz current administration.

[0020] Figures 11A and 11B show ATP content of cells incubated with control substances and glutamate, as described in Example 8.

[0021] Figures 12A-12H show ATP content of cells incubated with test compounds from Table 18 for 24 hours prior to addition of glutamate, as described in Example 8.

[0022] Figures 13A and 13B show ATP content of cells incubated with control substances and AMPA, as described in Example 8.

[0023] Figures 14A-14H show ATP content of cells incubated with test compounds from Table 18 for 24 hours prior to addition of AMPA, as described in Example 8.

[0024] Figures 15A-15L show calcium release from cells incubated with control substances and various concentrations of AMPA, as described in Example 8.

[0025] Figure 16 shows calcium release from cells incubated with control substances and glutamate, as described in Example 8.

[0026] Figures 17A-17H show calcium release from cells incubated with test compounds from Table 18 for 24 hours prior to addition of glutamate, as described in Example 8.

[0027] Figure 18 shows calcium release from cells incubated with control substances for 24 hours prior to addition of AMPA, as described in Example 8.

[0028] Figure 19 shows calcium release from cells incubated with test compounds from Table 18 for 24 hours prior to addition of APMA, as described in Example 8. DETAILED DESCRIPTION

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the arts to which the invention belongs. Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly 7   Attorney Docket No.01138-0045-00PCT used in the art. If there are a plurality of definitions for terms herein, those in this section prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sanghvi and Cook, American Chemical Society, Washington D.C., 1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and which is hereby incorporated by reference for any purpose. Where permitted, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can change, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0030] Before the present compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Definitions

[0031] “AMPAR” (or alternatively, “AMPA-R”), means the α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid glutamate receptor.

[0032] “Quality of life” means the extent to which a subject’s physical, psychological, and social functioning are impaired by a disease and / or treatment of a disease. Quality of life may be reduced in subjects having a seizure disorder.

[0033] “Slow the worsening of” and “slow worsening” mean to reduce the rate at which a medical condition moves towards an advanced state.

[0034] “Improves life expectancy” means to lengthen the life of a subject by treating one or more symptoms of a disease in the subject.

[0035] “Subject” means a human or non-human animal selected for treatment or therapy.

[0036] “Subject in need thereof” means a subject that is identified as in need of a therapy or treatment. 8   Attorney Docket No.01138-0045-00PCT

[0037] “Subject suspected of having” means a subject exhibiting one or more clinical indicators of a disease.

[0038] “Disease associated with AMPAR” means a disease or condition that is modulated by the activity of the AMPAR.

[0039] “Administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self- administering.

[0040] “Parenteral administration” means administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

[0041] “Subcutaneous administration” means administration just below the skin.

[0042] “Intravenous administration” means administration into a vein.

[0043] “Administered concomitantly” refers to the co-administration of two or more agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period and need not be coextensive.

[0044] “Duration” means the period during which an activity or event continues. In certain embodiments, the duration of treatment is the period during which doses of a pharmaceutical agent or pharmaceutical composition are administered.

[0045] “Therapy” means a disease treatment method. In certain embodiments, therapy includes, but is not limited to, administration of one or more pharmaceutical agents to a subject having a disease.

[0046] “Treat” means to apply one or more specific procedures used for the amelioration of at least one indicator of a disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutical agents.

[0047] “Ameliorate” means to lessen the severity of at least one indicator of a condition or disease. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

[0048] “At risk for developing” means the state in which a subject is predisposed to developing a condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not 9   Attorney Docket No.01138-0045-00PCT exhibit a sufficient number of symptoms to be diagnosed with the condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser extent required to be diagnosed with the condition or disease.

[0049] “Prevent the onset of” means to prevent the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

[0050] “Delay the onset of” means to delay the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

[0051] “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In certain embodiments, a dose is administered as a slow infusion.

[0052] “Dosage unit” means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted oligonucleotide.

[0053] “Therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.

[0054] “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent. For example, a pharmaceutical composition may comprise a sterile aqueous solution.

[0055] “Pharmaceutical agent” means a substance that provides a therapeutic effect when administered to a subject.

[0056] “Active pharmaceutical ingredient” means the substance in a pharmaceutical composition that provides a desired effect.

[0057] “Pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of a compound provided herein, i.e., a salt that retains the desired biological activity of the compound and does not have undesired toxicological effects when administered 10   Attorney Docket No.01138-0045-00PCT to a subject. Nonlimiting exemplary pharmaceutically acceptable salts of compounds provided herein include sodium and potassium salt forms. The terms “compound,” “oligonucleotide,” and “modified oligonucleotide” as used herein include pharmaceutically acceptable salts thereof unless specifically indicated otherwise.

[0058] “Saline solution” means a solution of sodium chloride in water.

[0059] “Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.

[0060] “Side effect” means a physiological response attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, kidney function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. Such side effects may be detected directly or indirectly. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

[0061] The term “blood” as used herein, encompasses whole blood and blood fractions, such as serum and plasma.

[0062] “Anti-miR” means an oligonucleotide having a nucleobase sequence complementary to a microRNA. In certain embodiments, an anti-miR is a modified oligonucleotide.

[0063] “Anti-miR-17” means a modified oligonucleotide having a nucleobase sequence at least 80%, at least 85%, at least 90%, or at least 95% complementary to miR-17.

[0064] “miR-17” means the mature miRNA having the nucleobase sequence 5’- CAAAGUGCUUACAGUGCAGGUAG-3’ (SEQ ID NO: 1).

[0065] “Target nucleic acid” means a nucleic acid to which an oligomeric compound is designed to hybridize.

[0066] “Targeting” means the process of design and selection of nucleobase sequence that will hybridize to a target nucleic acid.

[0067] “Targeted to” means having a nucleobase sequence that will allow hybridization to a target nucleic acid.

[0068] “Modulation" means a perturbation of function, amount, or activity. In certain embodiments, modulation means an increase in function, amount, or activity. In certain embodiments, modulation means a decrease in function, amount, or activity.

[0069] “Expression” means any functions and steps by which a gene’s coded information is converted into structures present and operating in a cell. 11   Attorney Docket No.01138-0045-00PCT

[0070] “Nucleobase sequence” means the order of contiguous nucleobases in an oligomeric compound or nucleic acid, typically listed in a 5’ to 3’ orientation, and independent of any sugar, linkage, and / or nucleobase modification.

[0071] “Contiguous nucleobases” means nucleobases immediately adjacent to each other in a nucleic acid.

[0072] “Nucleobase complementarity” means the ability of two nucleobases to pair non- covalently via hydrogen bonding.

[0073] “Complementary” means that one nucleic acid is capable of hybridizing to another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide capable of hybridizing to a target nucleic acid.

[0074] “Fully complementary” means each nucleobase of an oligonucleotide is capable of pairing with a nucleobase at each corresponding position in a target nucleic acid. In certain embodiments, an oligonucleotide is fully complementary (also referred to as 100% complementary) to a microRNA, i.e. each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. A modified oligonucleotide may be fully complementary to a microRNA, and have a number of linked nucleosides that is less than the length of the microRNA. For example, an oligonucleotide with 16 linked nucleosides, where each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in a microRNA, is fully complementary to the microRNA. In certain embodiments, an oligonucleotide wherein each nucleobase has complementarity to a nucleobase within a region of a microRNA stem-loop sequence is fully complementary to the microRNA stem-loop sequence.

[0075] “Percent complementarity” means the percentage of nucleobases of an oligonucleotide that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligonucleotide that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

[0076] “Percent identity” means the number of nucleobases in a first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid. In certain embodiments, the first nucleic acid is a microRNA and the second nucleic acid is a microRNA. In certain embodiments, the first nucleic acid is an oligonucleotide and the second nucleic acid is an oligonucleotide.

[0077] “Hybridize” means the annealing of complementary nucleic acids that occurs through nucleobase complementarity. 12   Attorney Docket No.01138-0045-00PCT

[0078] “Mismatch” means a nucleobase of a first nucleic acid that is not capable of Watson-Crick pairing with a nucleobase at a corresponding position of a second nucleic acid.

[0079] “Identical” in the context of nucleobase sequences, means having the same nucleobase sequence, independent of sugar, linkage, and / or nucleobase modifications and independent of the methylation state of any pyrimidines present.

[0080] “MicroRNA” means an endogenous non-coding RNA between 18 and 25 nucleobases in length, which is the product of cleavage of a pre-microRNA by the enzyme Dicer. Examples of mature microRNAs are found in the microRNA database known as miRBase (microrna.sanger.ac.uk / ). In certain embodiments, microRNA is abbreviated as “miR.”

[0081] “Oligomeric compound” means a compound that comprises a plurality of linked monomeric subunits. Oligomeric compounds include oligonucleotides.

[0082] “Oligonucleotide” means a compound comprising a plurality of linked nucleosides, each of which can be modified or unmodified, independent from one another.

[0083] “Naturally occurring internucleoside linkage” means a 3’ to 5’ phosphodiester linkage between nucleosides.

[0084] “Natural sugar” means a sugar found in DNA (2’-H) or RNA (2’-OH).

[0085] “Internucleoside linkage” means a covalent linkage between adjacent nucleosides.

[0086] “Linked nucleosides” means nucleosides joined by a covalent linkage.

[0087] “Nucleobase” means a heterocyclic moiety capable of non-covalently pairing with another nucleobase.

[0088] “Nucleoside” means a nucleobase linked to a sugar moiety.

[0089] “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of a nucleoside.

[0090] “Compound comprising a modified oligonucleotide consisting of” a number of linked nucleosides means a compound that includes a modified oligonucleotide having the specified number of linked nucleosides. Thus, the compound may include additional substituents or conjugates. Unless otherwise indicated, the modified oligonucleotide is not hybridized to a complementary strand and the compound does not include any additional nucleosides beyond those of the modified oligonucleotide.

[0091] “Modified oligonucleotide” means a single-stranded oligonucleotide having one or more modifications relative to a naturally occurring terminus, sugar, nucleobase, and / or internucleoside linkage. A modified oligonucleotide may comprise unmodified nucleosides. 13   Attorney Docket No.01138-0045-00PCT

[0092] “Modified nucleoside” means a nucleoside having any change from a naturally occurring nucleoside. A modified nucleoside may have a modified sugar and an unmodified nucleobase. A modified nucleoside may have a modified sugar and a modified nucleobase. A modified nucleoside may have a natural sugar and a modified nucleobase. In certain embodiments, a modified nucleoside is a bicyclic nucleoside. In certain embodiments, a modified nucleoside is a non-bicyclic nucleoside.

[0093] “Modified internucleoside linkage” means any change from a naturally occurring internucleoside linkage.

[0094] “Phosphorothioate internucleoside linkage” means a linkage between nucleosides where one of the non-bridging atoms is a sulfur atom.

[0095] “Modified sugar moiety” means substitution and / or any change from a natural sugar.

[0096] “Unmodified nucleobase" means the naturally occurring heterocyclic bases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

[0097] “5-methylcytosine” means a cytosine comprising a methyl group attached to the 5 position.

[0098] “Non-methylated cytosine” means a cytosine that does not have a methyl group attached to the 5 position.

[0099] “Modified nucleobase” means any nucleobase that is not an unmodified nucleobase. [000100] “Sugar moiety” means a naturally occurring furanosyl or a modified sugar moiety. [000101] “Modified sugar moiety” means a substituted sugar moiety or a sugar surrogate. [000102] “2’-O-methyl sugar” or “2’-OMe sugar” means a sugar having an O-methyl modification at the 2’ position. [000103] “2’-O-methoxyethyl sugar” or “2’-MOE sugar” means a sugar having an O- methoxyethyl modification at the 2’ position. [000104] “2’-fluoro” or “2’-F” means a sugar having a fluoro modification of the 2’ position. [000105] “Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including by not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments, the 4 to 14   Attorney Docket No.01138-0045-00PCT 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2’-carbon and the 4’-carbon of the furanosyl. Nonlimiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt. [000106] “Locked nucleic acid (LNA) sugar moiety” means a substituted sugar moiety comprising a (CH2)-O bridge between the 4’ and 2’ furanose ring atoms.  [000107] “ENA sugar moiety” means a substituted sugar moiety comprising a (CH2)2-O bridge between the 4’ and 2’ furanose ring atoms.   [000108] “Constrained ethyl (cEt) sugar moiety” means a substituted sugar moiety comprising a CH(CH3)-O bridge between the 4' and the 2' furanose ring atoms. In certain embodiments, the CH(CH3)-O bridge is constrained in the S orientation. In certain embodiments, the CH(CH3)-O is constrained in the R orientation.  [000109] “S-cEt sugar moiety” means a substituted sugar moiety comprising an S- constrained CH(CH3)-O bridge between the 4' and the 2' furanose ring atoms.  [000110] “R-cEt sugar moiety” means a substituted sugar moiety comprising an R- constrained CH(CH3)-O bridge between the 4' and the 2' furanose ring atoms.  [000111] “2’-O-methyl nucleoside” means a 2’-modified nucleoside having a 2’-O-methyl sugar modification. [000112] “2’-O-methoxyethyl nucleoside” means a 2’-modified nucleoside having a 2’-O- methoxyethyl sugar modification. A 2’-O-methoxyethyl nucleoside may comprise a modified or unmodified nucleobase. [000113] “2’-fluoro nucleoside” means a 2’-modified nucleoside having a 2’-fluoro sugar modification. A 2’-fluoro nucleoside may comprise a modified or unmodified nucleobase. [000114] “Bicyclic nucleoside” means a 2’-modified nucleoside having a bicyclic sugar moiety. A bicyclic nucleoside may have a modified or unmodified nucleobase. [000115] “cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEt nucleoside may comprise a modified or unmodified nucleobase. [000116] “S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety. [000117] “R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety. [000118] “β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside. [000119] “β-D-ribonucleoside” means a naturally occurring RNA nucleoside. [000120] “LNA nucleoside” means a nucleoside comprising a LNA sugar moiety. [000121] “ENA nucleoside” means a nucleoside comprising an ENA sugar moiety. [000122] “Hydrogen bond acceptor” means the component of a hydrogen bond that does not supply the shared hydrogen atom. 15   Attorney Docket No.01138-0045-00PCT [000123] “Hydrogen bond donor” means the bond or molecule that supplies the hydrogen atom of a hydrogen bond. Overview [000124] Anti-miR-17 compounds were previously discovered to be useful in treating polycystic kidney disease (PKD). Through screening of a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties, the compound RGLS4326 (or RG4326), was identified as a candidate to be pursued for clinical development for treatment of PKD in humans. The compound RG4326, methods of making the compound, in vitro and in vivo data demonstrating its efficacy in models of PKD, and metabolic stability and pharmacokinetic and safety profile are described in WO 2018 / 106566 A1, the entire contents of which are hereby incorporated by reference. [000125] Following pursuit of clinical development and consultation with FDA, it was indicated that dose / duration limitations for RG4326 could be well below 1 mg / kg for long-term chronic dosing. However, after clinical trials were initiated to study RG4326 in humans for use in treating PKD, CNS-related findings were observed at high doses of RG4326 in nonclinical toxicology mouse models. RG4326 was found to be an antagonist of AMPAR, a glutamate receptor and ion channel on excitatory synapses in the central nervous system (CNS). AMPAR mediates fast excitatory neurotransmission and is a key component of all neuronal networks. [000126] It has now been surprisingly discovered that AMPAR antagonist activities of the compounds provided herein, including the compounds in Table 1, which previously rendered them undesirable candidates for advancement in PKD pharmaceutical development, are beneficial for advancing these and other compounds in clinical development for use in the treatment of nervous system disorders including epilepsy. [000127] It has been discovered that the compounds provided herein, including the compounds in Table 1, are competitive inhibitors of AMPAR, and are therefore useful for treating nervous system disorders. Compounds [000128] Provided herein is a compound comprising a modified oligonucleotide consisting of 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, or 4-15 linked nucleosides, or a pharmaceutically acceptable salt thereof. In certain embodiments, the modified oligonucleotide consists of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides. [000129] In certain embodiments, the last four nucleosides at the 3’ end of the modified oligonucleotide have the nucleobase sequence UUUG. 16   Attorney Docket No.01138-0045-00PCT [000130] In certain embodiments, at least two of the last four nucleosides at the 3’ end of the modified oligonucleotide are bicyclic nucleosides. In certain embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleosides of the modified oligonucleotide are bicyclic nucleosides. In certain embodiments, each bicyclic nucleoside is independently selected from S-cEt nucleoside, LNA nucleoside, and ENA nucleoside. [000131] In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. [000132] In certain embodiments, the modified oligonucleotide consists of 4 linked nucleosides. In certain embodiments, the modified oligonucleotide is UUUG. In some embodiments, the modified oligonucleotide is UFUMUSGSor USUSUSGS, wherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides. [000133] In certain embodiments, the modified oligonucleotide consists of 9 linked nucleosides. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5’-AGCACUUUG-3’, wherein each cytosine is independently selected from a non- methylated cytosine and a 5-methylcytosine. [000134] In certain embodiments, the modified oligonucleotide has the following nucleoside pattern in the 5’ to 3’ orientation: NSNSNMNFNFNFNMNSNS wherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides. [000135] In certain embodiments, the compound consists of the modified oligonucleotide. [000136] In certain embodiments, the pharmaceutically acceptable salt is a sodium salt. [000137] In certain embodiments, the modified oligonucleotide is RG4326, and has the following sequence and chemical modification pattern: ASGSCMAFCFUFUMUSGS where nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides, each cytosine is a non-methylated cytosine, and all linkages are phosphorothioate linkages. [000138] RG4326 can be further described as having the structure: 17   Attorney Docket No.01138-0045-00PCT[000139] Provided herein is a modified oligonucleotide having the structure: 18   Attorney Docket No.01138-0045-00PCT[000140] In some embodiments, a pharmaceutically acceptable salt of a modified oligonucleotide comprises fewer cationic counterions (such as Na+) than there are phosphorothioate and / or phosphodiester linkages per molecule (i.e., some phosphorothioate and / or phosphodiester linkages are protonated). In some embodiments, a pharmaceutically acceptable salt of a modified oligonucleotide comprises fewer than 8 cationic counterions (such as Na+) per molecule of modified oligonucleotide. That is, in some embodiments, a pharmaceutically acceptable salt of the modified oligonucleotide may comprise, on average, 1, 2, 3, 4, 5, 6, or 7 cationic counterions per molecule of modified oligonucleotide, with the remaining phosphorothioate groups being protonated. [000141] Table 1 below shows certain nonlimiting exemplary compounds provided herein: 19   Attorney Docket No.01138-0045-00PCT Table 1: Certain CompoundsMethods of Use [000142] Methods of treating a nervous system disorder in a subject are provided, comprising administering to the subject a therapeutically-effective amount of a compound provided herein, including the compounds in Table 1, comprising a modified oligonucleotide that binds to and inhibits AMPAR, or a pharmaceutically acceptable salt thereof. In some embodiments, the nervous system disorder is epilepsy. [000143] In some embodiments, the nervous system disorder is pharmacoresistant epilepsy. In other embodiments, the nervous system disorder is a non-pharmacoresistant epilepsy. In some embodiments, the compound provided herein is used as a 1st-line therapy. [000144] In some embodiments, the compound provided herein is used to treat a central or peripheral nervous system disorder in a subject. In some embodiments, the nervous system disorder is hearing loss, motor disease, amyotrophic lateral sclerosis (ALS), pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke or glioblastoma. [000145] In some embodiments, the compound provided herein is formulated in a pharmaceutical composition with one or more pharmaceutically-acceptable diluents or 20   Attorney Docket No.01138-0045-00PCT excipients. In some embodiments, the compound provided herein is formulated in a saline solution. In some embodiments, the pharmaceutical composition is a lyophilized composition. [000146] In some embodiments, the compound provided herein is any one of the compounds set forth in Table 1. [000147] In any of the embodiments provided herein, the subject is a human subject. In certain embodiments, the human subject is an adult. In certain embodiments, an adult is at least 18 years of age. In certain embodiments, the human subject is a pediatric subject, i.e. the subject is less than 18 years of age. Pediatric populations may be defined by regulatory agencies. In certain embodiments, the human subject is an adolescent. In certain embodiments, an adolescent is at least 12 years of age and less than 18 years of age. In certain embodiments, the human subject is a child. In certain embodiments, a child is at least two years of age and less than 12 years of age. In certain embodiments, the human subject is an infant. In certain embodiments, and infant is at least one month of age and less than two years of age. In certain embodiments, the subject is a newborn. In certain embodiments, a newborn is less than one month of age. [000148] Any of the compounds described herein may be for use in therapy. [000149] Any of the compounds provided herein may be for use in the preparation of a medicament. Any of the compounds provided herein may be for use in the preparation of a medicament for the treatment of a nervous system disorder, preferably epilepsy. [000150] Any of the modified oligonucleotides provided herein may be for use in the preparation of a medicament. Any of the modified oligonucleotides provided herein may be for use in the preparation of a medicament for the treatment of a nervous system disorder. [000151] Any of the pharmaceutical compositions provided herein may be for use in the treatment of a nervous system disorder. Certain Additional Therapies [000152] Treatments for a nervous system disorder or any of the conditions listed herein may comprise more than one therapy. As such, in certain embodiments, provided herein are methods for treating a subject having or suspected of having a nervous system disorder comprising administering at least one therapy in addition to administering compound provided herein which mediates AMPAR. [000153] In certain embodiments, the at least one additional therapy comprises a pharmaceutical agent. In certain embodiments, the additional pharmaceutical agent is an anti- epileptic drug (AED). In certain embodiments, the AED is selected from carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, everolimus, gabapentin, lacosamide, 21   Attorney Docket No.01138-0045-00PCT lamotrigine, levetiracetam, oxcarbazepine, perampanel, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin and zonisamide. Certain Pharmaceutical Compositions [000154] Provided herein are pharmaceutical compositions comprising a compound or modified oligonucleotide provided herein, and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In certain embodiments, the aqueous solution is a saline solution. As used herein, pharmaceutically acceptable diluents are understood to be sterile diluents. Suitable administration routes include, without limitation, intravenous and subcutaneous administration. In certain embodiments, administration is intravenous administration. In certain embodiments, administration is subcutaneous administration. In certain embodiments, administration is oral administration. [000155] In certain embodiments, a pharmaceutical composition is administered in the form of a dosage unit. For example, in certain embodiments, a dosage unit is in the form of a tablet, capsule, or a bolus injection. [000156] In certain embodiments, a pharmaceutical agent is a modified oligonucleotide which has been prepared in a suitable diluent, adjusted to Ph 7.0-9.0 with acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified oligonucleotide is subsequently reconstituted with a suitable diluent, e.g., aqueous solution, such as water or physiologically compatible buffers such as saline solution, Hanks’s solution, or Ringer’s solution. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion. The lyophilized drug product may be packaged in a 2 Ml Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal. [000157] In certain embodiments, the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents. [000158] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers; such additional materials also include, 22   Attorney Docket No.01138-0045-00PCT but are not limited to, excipients such as alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In various embodiments, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions. [000159] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In one method, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In another method, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.  [000160] In certain embodiments, a pharmaceutical composition provided herein comprise a polyamine compound or a lipid moiety complexed with a nucleic acid. In certain embodiments, such preparations comprise one or more compounds each individually having a structure defined by formula (Z) or a pharmaceutically acceptable salt thereof,  23   Attorney Docket No.01138-0045-00PCT wherein each Xaand Xb, for each occurrence, is independently C1-6 alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least n + 2 of the R moieties in at least about 80% of the molecules of the compound of formula (Z) in the preparation are not H; m is 1, 2, 3 or 4; Y is O, NR2, or S; R1is alkyl, alkenyl, or alkynyl; each of which is optionally substituted with one or more substituents; and R2is H, alkyl, alkenyl, or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n = 0, then at least n + 3 of the R moieties are not H. Such preparations are described in PCT publication WO / 2008 / 042973, which is herein incorporated by reference in its entirety for the disclosure of lipid preparations. Certain additional preparations are described in Akinc et al., Nature Biotechnology 26, 561 – 569 (01 May 2008), which is herein incorporated by reference in its entirety for the disclosure of lipid preparations.   [000161] In certain embodiments, a pharmaceutical composition provided herein is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. [000162] In certain embodiments, a pharmaceutical composition provided herein is a solid (e.g., a powder, tablet, and / or capsule). In certain of such embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents. [000163] In certain embodiments, a pharmaceutical composition provided herein is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [000164] In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used. [000165] In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in 24   Attorney Docket No.01138-0045-00PCT certain embodiments, pharmaceutical compositions include liposomes coated with a tissue- specific antibody. [000166] In certain embodiments, a pharmaceutical composition provided herein comprises a sustained-release system. A non-limiting example of such a sustained-release system is a semi- permeable matrix of solid hydrophobic polymers. In certain embodiments, sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months. [000167] Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. [000168] In certain embodiments, a pharmaceutical composition provided herein comprises a modified oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. [000169] In certain embodiments, one or more modified oligonucleotides provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances, the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.   [000170] In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound 25   Attorney Docket No.01138-0045-00PCT is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392). [000171] Additional administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the kidney). Certain Kits  [000172] The present invention also provides kits. In some embodiments, the kits comprise one or more compounds comprising a modified oligonucleotide disclosed herein. In some embodiments, the kits may be used for administration of the compound to a subject. [000173] In certain embodiments, the kit comprises a pharmaceutical composition ready for administration. In certain embodiments, the pharmaceutical composition is present within a vial. A plurality of vials, such as 10, can be present in, for example, dispensing packs. In some embodiments, the vial is manufactured so as to be accessible with a syringe. The kit can also contain instructions for using the compounds. [000174] In some embodiments, the kit comprises a pharmaceutical composition present in a pre-filled syringe (such as a single-dose syringes with, for example, a 27 gauge, ½ inch needle with a needle guard), rather than in a vial. A plurality of pre-filled syringes, such as 10, can be present in, for example, dispensing packs. The kit can also contain instructions for administering the compounds comprising a modified oligonucleotide disclosed herein. [000175] In some embodiments, the kit comprised a modified oligonucleotide provided herein as a lyophilized drug product, and a pharmaceutically acceptable diluent. In preparation for administration to a subject, the lyophilized drug product is reconstituted in the pharmaceutically acceptable diluent. [000176] In some embodiments, in addition to compounds comprising a modified oligonucleotide disclosed herein, the kit can further comprise one or more of the following: syringe, alcohol swab, cotton ball, and / or gauze pad. EXAMPLES [000177] The following examples are presented to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. 26   Attorney Docket No.01138-0045-00PCT [000178] Those of ordinary skill in the art will readily adopt the underlying principles of this discovery to design various compounds without departing from the spirit of the current invention. Abbreviations [000179] The following abbreviations may be useful herein when considering the following Examples: Table 2: AbbreviationsExample 1 Example 1A: AMPAR binding [000180] The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct- derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous 27   Attorney Docket No.01138-0045-00PCT administration. A phase 1 single ascending dose (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a phase 1 multiple ascending dose (MAD) clinical trial in healthy volunteers that was initiated in May 2018. A phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) was initiated in October 2020. [000181] Subsequent to the initiation of the phase 1 MAD clinical trial, nonclinical toxicology studies revealed central nervous system (CNS)-related findings, including abnormal gait, reduced motor activity, and / or prostration, at high doses of RGLS4326. To identify potential candidates for off-target pharmacology, a panel of 174 targets including G-protein coupled receptors, transporters, ion channels, nuclear receptors, and cytokine receptors was evaluated in vitro for possible interactions with RGLS4326. RGLS4326 was found to be an antagonist of the AMPA glutamate receptor, with a 50% inhibitory concentration (IC50) of 4.6 uM (14.2 ug / mL) based on ligand binding and a functional IC50of 300-600 nM (0.9-1.8 ug / mL) based on patch clamp activity. AMPA receptors are ion channels on excitatory synapses in the CNS that mediate fast excitatory neurotransmission and, therefore, are key components of all neuronal networks. Such an interaction with the AMPA receptor could explain the CNS- mediated findings observed at high doses of RGLS4326 in nonclinical toxicology models. Example 1B: Compounds having AMPAR Antagonist Activity [000182] The activity of anti-miR-17 compounds was evaluated in a radioligand binding assay which measured the binding of the [3H] AMPA ligand to the AMPAR present on rat brain synaptic membranes, in the presence of increasing concentrations of anti-miR-17 compound. anti-miR-17 compounds with affinity for the AMPAR will bind to and compete with the binding of the [3H] AMPA ligand. [000183] The assay was performed according to previously published methods (Honore et al., J Neurochem., 1982, 38(1):173-178; Olsen et al., Brain Res., 1987, 402(2):243-254).5.0 nM of the ligand [3H] AMPA, 1.0 mM of the non-specific ligand L-Glutamic acid, and anti-miR compound at uM concentrations were incubated with synaptic membranes prepared from Wistar rat cerebral cortex for 90 minutes. The compounds shown in Table 2 were tested in three experiments. Anti-miRs targeted to microRNAs other than miR-17 were used as control compounds (RG5124 targeted to miR-33a; RG5365 targeted to let-7a; RG8093 targeted to miR- 214). RGLS4326 and RG-NG-1001 were also tested in each experiment, as it was demonstrated to bind to and inhibit the activity of the AMPAR. The amount of the [3H] AMPA ligand was 28   Attorney Docket No.01138-0045-00PCT quantitated by radioligand binding, and is shown in Tables 3, 4, and 5. As illustrated by the data, the compounds vary in their ability to inhibit binding of the radiolabeled ligand to the AMPAR. Table 3: Inhibition of Ligand Binding to AMPAR Experiment #1Table 4: Inhibition of Ligand Binding to AMPAR Experiment #2Table 5: Inhibition of Ligand Binding to AMPAR Experiment #3[000184] Based on data obtained with anti-miR-17 compounds in radioligand binding assays as shown above in Tables 3-5, AMPAR binding was additionally profiled using a series of shortened length sequences (i.e., 4-nucleotide “tetramers”). In these studies, RGLS4326 was included as a positive control for validated AMPA-R binding; RG-NH-1026 was included as a 29   Attorney Docket No.01138-0045-00PCT negative control for validated no AMPAR binding. As shown in Table 6, RG8524 and RG8525 demonstrated ability to inhibit binding of radiolabeled ligand to the AMPAR, with RG8525 meeting criteria for significance in the assay (≥ 50% inhibition; N.C. = not calculated for not meeting IC50 criteria). Table 6: Inhibition of Ligand Binding to AMPAR Experiment #4[000185] To evaluate functional antagonism of anti-miR-17 oligonucleotides towards the AMPAR, certain oligonucleotides were tested using the manual whole-cell patch clamp technique, which records membrane currents as a measure of AMPAR activity. [000186] Manual whole-cell patch clamp studies were performed by Metrion Biosciences (Cambridge, UK). Whole-cell voltage clamp experiments were performed at room temperature (18 - 21 °C) using an EPC10 patch clamp amplifier using Patchmaster software (HEKA Elektronik). Glass patch pipettes were fabricated from borosilicate glass capillaries (Harvard Apparatus) to resistances between 1.4 and 2.5 MΩ. Membrane currents were recorded using the whole-cell patch clamp technique. ChanTest® GluA1 / GluA4 EZCells were clamped at a holding potential of -80 mV and membrane currents elicited by 10 μM (S)-AMPA delivered using a VC38 perfusion system (ALA Scientific Instruments). The minimal current amplitude values were measured with each application of 10 μM (S)-AMPA. The fractional change of current amplitude produced by each concentration of compound was calculated relative to the control current (pre-compound) and expressed as percentage change (% inhibition) for each cell. The compounds tested are shown in Table 7. Table 7: Functional Antagonism of AMPAR in Whole-Cell Patch Clamp Studies30   Attorney Docket No.01138-0045-00PCT[000187] It was discovered that RG4326 inhibits [3H] AMPA ligand binding to synaptic membranes from the rat cerebral cortex and that RG4326 inhibits whole-cell patch-clamp studies in HEK293 cells overexpressing rGluA1 and rGluA2 in response to 3 mM glutamate. [000188] Accordingly, RGLS4326 was found to be an antagonist of the AMPAR, with a 50% inhibitory concentration (IC50) of 4.6 uM (14.2 ug / mL) based on ligand binding and a functional IC50of 300-600 nM (0.9-1.8 ug / mL) based on patch clamp activity. Example 2 Maximum Tolerated Dose (MTD) Pilot Study and Comparative Dose Assessment [000189] Compounds were evaluated in maximum tolerated dose (MTD and pentylenetetrazole (PTZ) seizure models. The experiment was conducted in two parts, i.e.: the first part (Example 2A) was a maximum tolerated dose (MTD) dose pilot study of RG4047 and RG4326 at 4 dose-levels each (32 mice), with including PTZ dose finding (16 mice); and the second part (Example 2B), was a PTZ study of RG4047 and RG4326 at 2 dose-levels each (90 mice). A negative control was included in both parts of the study (a non-AMPAR binding compound), and reference group was perampanel. [000190] For each study, C57Bl / 6J male mice (Jackson Laboratories), age 6-7 weeks, were used. The mice were assigned randomly to treatment groups, and the study was blinded. Animals were allowed to acclimate for no less than 5 days and housed on a 12 hr light / dark cycle (lights on 7:00 AM). No more than 4 mice were house in each cage, i.e., a ventilated cage rack system; diet consisted of standard rodent chow and water ad libitum. Example 2A – MTD Pilot Study [000191] The following parameters were used for this study: 1. Route(s) of administration: ICV dosing of RG4047, RG4326; IP dosing of PTZ 2. Dose Volume(s): 4 ul for RG compounds, 5ml / kg (PTZ) 3. Formulation(s): vehicle, Ca2+and Mg2+free dPBS 4. Dose Levels: PTZ tested at 60, 65, and 75 mg / kg 5. Dose Frequency: Once 6. Study duration: 8 Days 7. Number of Groups: 14 31   Attorney Docket No.01138-0045-00PCT 8. Number of animals per group: Groups 1-12 (4 each group), Groups 13-14 (8 each) 9. Total number of animals: 64 [000192] For the ICV administration, mice were anesthetized and positioned for injections. The skin over the skull was incised, and a small hole was made in the skull above the target using a microdrill. The stereotactic coordinates were anteroposterior (AP), −0.4 mm; mediolateral (ML), + / - 1.0-1.5 mm; dorsoventral (DV), −3.0 mm from the bregma for injection into both the right and left lateral cerebral ventricles (Hironaka et al, 2015). Animals were injected unilaterally with 4 μl into the right lateral cerebral ventricle. Compounds were injected over 1-2 min, and the needle was left in place for 0.5-1 min prior to withdrawal. The incision was closed with sutures, wound clips, or VetBond. [000193] The design of this study is further illustrated below in Tables 8A and 8B. 32   Attorney Docket No.01138-0045-00PCT Table 8A: MTD Study Design – AMPAR Inhibitors33   Attorney Docket No.01138-0045-00PCT Table 8B: MTD Study Design – PTZ Dosing[000194] For groups 1-12, following ICV treatment on Day 0, animals were monitored for 7 days in which daily health checks, body weight, and mortality was recorded. On Day 7, brain and kidney were collected and fixed (10% formalin) and stored pending histology. [000195] For groups 13-14, mice were dosed with vehicle into right lateral cerebral ventricle on Day 0, and 24 hr post ICV administration, injections of PTZ were made at a volume of 5 ml / kg to produce a final dose. Immediately following PTZ administration, animals were observed for 30 mins. where times to initial myoclonic and tonic hindlimb extensor response were measured. A 30 min. maximum latency to seizure period was imposed. [000196] Racine Scale: Clinical behavior was scored using the following adapted Racine scale: Score 0, no seizures observed; Score 1, Behavioral arrest (freezing, fixed gaze, staring), vibrissae twitching, mouth or facial clonus Score 2, Pop or jump, straub tail, foot splay, head nodding Score 3, Forelimb clonus, extended forelimbs, multiple pops or jerks Score 4, Rearing and bilateral forelimb clonus, Running and running / bouncing 34   Attorney Docket No.01138-0045-00PCT Score 5, All limb clonus, rearing and falling (stay fallen on rear side), Score 6: Tonic hindlimb extensions, cardiac arrest. [000197] Behavior was scored by an observer blind to treatment. The highest score reached during every 5 min during the 30 min after PTZ administered was recorded. [000198] The data was analyzed by one or two-way ANOVA, followed by Dunnett’s or Fisher’s LSD post-hoc tests to determine differences between treatments. Data are expressed as the average ± SEM. Comparison of the perampanel reference group to the vehicle group was analyzed by t-test. A p value of less than 0.05 was used to indicate statistical significance. [000199] The results from the MTS study with regard to groups 1-12 are shown below in Table 9. Negative control compound was well-tolerated at high doses (consistent with previous ICV studies). The MTD for RG4326 was predicted at ~2.5 < 5.0 ug ICV in the pilot study. Due to mortality observed at high doses for RG4326 and RG4047 (e.g., 600, 300, 100 ug), MTD dosing parameters were modified accordingly. RG4047 MTD was not attained, and was therefore likely to be under 2.5 ug. All animal deaths were reported to occur within the first 5-8 hrs post-ICV injection. Mice injected with 2.5 ug RG4326 were reported to display some immediate signs of respiratory distress and were provided heating pads. All animals were reported to fully recover on Day 2 of observation. Results from previous studies corroborate well-tolerated ICV doses of negative control compound. Based on these data, a top dose of 2.0 ug was selected for RG4326 in this model. Table 9: Summary Results from 7-day MTD Study35   Attorney Docket No.01138-0045-00PCT Example 3 PTZ Optimal Dose Pilot Study [000200] A pilot study was conducted to determine the optimal dose of PTZ to be used for this experiment, considering 60, 65, and 70 mg / kg PTZ (IP) . For this pilot study, after the three doses of PTZ were administered to the animals, they were observed using the Racine Scale at 5 min intervals up to 30 min. Behavior was scored by an observer blind to treatment. The highest score reached at each 5 min interval during the 30 min observation period was recorded. Immediately following PTZ injection (time 0), assessment was made for time from latency to onset of clonic seizure, and from there, the animals were monitored for signs of tonic seizure. Clinical behavior (Racine Scale) was recorded every 5 min for 30 min. [000201] Figures 1A and 1B show the latency to seizure data (clonic and tonic seizure, respectively), for the three dosages of 60 mpk, 65mpk and 70 mpk. As shown in Figures 1A and 1B, PTZ at all doses induced rapid onset of clonic (Fig.1A) and tonic (Fig.1B), seizures. However, mortality rates (indicated in Figure 1B), reflected an increased trend in mortality (3 / 6) with the higher dose (70 mpk). By comparison, no deaths were reported at the lowest tested dose of 60 mpk. [000202] Figure 2 shows the Racine score over time following PTZ administration in this experiment. As shown on Figure 2, the lowest dose of PTZ at 60 mpk was capable of inducing clonic / tonic seizure, but seizure intensity quickly dissipated after an initial 5 min post-PTZ. Conversely, 70 mpk PTZ evoked higher average Racine scores, consistent with the desired outcome of an increased tonic seizure incidence in the model. [000203] Figure 3 is a bar graph showing survival data in this experiment. Following 30 min post-treatment of PTZ, final survival data comparing the effect of the 60 mpk, 65 mpk and 70 mpk dosages revealed diminished survival rates at 70 mpk of 4 / 6 injected mice, as compared with 6 / 6 for 60 mpk, and 4 / 5 for 65 mpk. [000204] From this data, a PTZ dose of 70 mpk was selected as an appropriate working concentration for inducing sufficient seizure profiles and increasing mortality risk in the model. Higher doses of PTZ (e.g., 80 mpk, 100 mpk) were profiled in earlier studies and found to exert excessively strong effects on seizure latency times, Racine scoring and most importantly 30 min survival. 36   Attorney Docket No.01138-0045-00PCT Example 4 PTZ Study of RG4326 Compared with Perampanel [000205] CNS effects for the test compound RG4326 was compared to negative control compound, vehicle, and perampanel. Seizures were induced in study mice by administering PTZ using the dose of 70 mpk, as determined in Example 3. The groups of mice were pretreated with RG4326, NCC, and perampanel (or received no treatment in the vehicle group), prior to the PTZ administration, and then data was collected regarding the effects of the study drugs (as compared to vehicle), in addressing the CNS effects from the PTZ-induced seizures. Results from this PTZ study demonstrated a significant anti-seizure effect of RG4326 across all major study endpoints, including seizure and cardiac arrest latency times, Racine scale scoring, and overall survival. [000206] Using the PTZ dose of 70 mpk as determined in Example 3, the CNS effects via Racine scoring for RG4326, as compared with perampanel or negative control compound (NCC) were evaluated using the following parameters: 1. Route(s) of administration: ICV RG4326 and NCC; PTZ (IP); Perampanel (PO) 2. Dose Volume(s): 10 ml / kg (Perampanel), 4 uL for test articles, 5ml / kg (PTZ) 3. Formulation(s): dPBS; PER vehicle saline 4. Dose Levels: Perampanel, 2 mg / kg; PTZ, 70 mg / kg 5. Dose Frequency: Once 6. Study duration: 2 days 7. Number of animals per group: 15 8. Number of groups: 8 9. Total number of animals: 120 [000207] The design of this study is further illustrated below in Table 10. As reflected in Table 10, study drugs RG4326 and NCC were administered to all mice in groups 2-7 via ICV administration twenty-four hours prior to PTZ administration at the dosages indicated in Table 10. Group 1 received no pre-treatment, only the sterile vehicle PBS. Group 8 received approximately 2 mg / kg of perampanel, as a reference compound, 30 minutes prior to PTZ administration by mouth (PO). The ICV administration for the test drugs proceeded as described above in Example 2A. [000208] To assess the CNS effects, mice were first acclimated to the procedure room for at least 30 minutes. Pentylenetetrazole (PTZ; Sigma Aldrich) was formulated in water to a concentration of 16 mg / ml. Injections of PTZ were made at a volume of 5 ml / kg to produce a final dose of 70 mg / kg (IP), to induce seizure conditions. Immediately following PTZ 37   Attorney Docket No.01138-0045-00PCT administration, animals were observed for 30 minutes, and latency of clonic and tonic hindlimb extensor responses were recorded, as well as Racine Scale scoring, to compare the effect of the RG4326, NCC, and perampanel on PTZ-induced seizures. Table 10. PTZ Study Design[000209] Latency to Tonic Seizure. The comparative latency to tonic seizure data obtained from this study is reported in Figure 4. Figure 4 shows the mean duration (in seconds), of the period from latency to onset of tonic seizure for all groups. As demonstrated in Figure 4, animals given perampanel (reference compound) at a concentration of 2 mg / kg showed a significant increase in duration from latency to tonic seizure when compared to vehicle treated animals (**** P <0.0005, 1-w ANOVA, Dunnett’s). RG4326 treated groups showed significance (*P <0.05) at 0.25 and 0.5 ug, as compared to vehicle. Despite a trend toward increased latency times for additional treatment conditions, all other groups showed no significant difference, when compared to vehicle-treated animals, for the time span from latency to onset of tonic seizure. [000210] Latency to Cardiac Arrest. Comparative latency to cardiac arrest (e.g., death), data from this study is reported in Figure 5. Figure 5 reports the mean column bar graph of the duration (in seconds), from latency to tonic cardiac arrest for all groups. As demonstrated by Figure 5, animals given perampanel (reference compound) at a concentration of 2 mg / kg showed 38   Attorney Docket No.01138-0045-00PCT a significant increase in the time span from latency to tonic cardiac arrest when compared to vehicle treated animals (*** P <0.005, 1-w ANOVA, Dunnett’s). RG4326 at 0.5 ug showed significance (* P <0.05). Although the other treatment groups reflected trends toward increased time spans from latency to cardiac arrest (as with latency to tonic seizure), the other treatment groups showed no significant difference when compared to vehicle treated animals for the latency to cardiac arrest duration. [000211] Racine scoring. The Racine scoring for all groups, as obtained in 5 min intervals up to 30 min post PTZ administration, is reported in the line graph of Figure 6. In collecting the Racine scores for Figure 6, all animals that had an endpoint of cardiac arrest were scored at the maximum score (6) for all remaining time points in the 30 min observation interval. Animals given perampanel (reference compound) at a concentration of 2 mg / kg showed a statistically significant reduction in Racine score at all time points post PTZ administration (* P <0.05, 2-w ANOVA, Dunnett’s), when compared to vehicle treated animals. RG4326 treated animals showed significant reduction in Racine score in the 0.5 ug treated group. No other groups showed significance at any of the time points when compared to vehicle treated animals. [000212] Mortality over time after PTZ administration. The deaths occurring for every group at each 5 min observation period were recorded for up to 30 min after the PTZ administration. The survival data is reported in the line graph of Figure 7. Tables 11 and 12, below, further report the total deaths (Table 11) and percent mortality rates based on starting sample size per cohort (Table 12), at each interval, and over time. As can be seen from this data, significant improvement in post-PTZ survival was evident for permapanel (reference compound) at 2 mpk (Group 8 in Tables 11 and 12, and on line graph), and RG4326 at 0.5 ug (Group 4 in Tables 11 and 12, and on graph). (** P <0.005, logrank, Mantel-Cox test). Table 11: Total Mortality Rates Over 30 min for Example 4 Cohorts39   Attorney Docket No.01138-0045-00PCTTable 12: Percent Mortality Rates Over 30 min for Example 4 Cohorts[000213] In summary, in the study of this Example 4, RG4326 was tested for its ability to confer protection against PTZ-induced seizures when administered 24 hrs by ICV injection, prior to PTZ administration. Based on pilot data seeking to establish an MTD for RG4326 in this model, doses of 2.0 ug, 1.0 ug, 0.5 ug and 0.25 ug were selected. Perampanel was selected as a reference compound and tested at 2 mg / kg (PO), based on published data for optimized efficacy in the PTZ model. Hanada et al, Epilepsia (2011) 52(7):1331-40. Results from this PTZ study demonstrate a significant anti-seizure effect of RG4326 (0.5 ug ICV treatment), with impact across all major study endpoints, including seizure and cardiac arrest latency times, Racine scale scoring, and overall survival. 40   Attorney Docket No.01138-0045-00PCT Example 5 MTD Study for Evaluation of RG4326 In 6-Hz Seizure Model [000214] Two MTD dose studies for RG4326 were conducted to assess the dose to use in evaluating the compound in 6-Hz induced seizure experiments (Examples 6 and 7), using two different mice models, i.e., C57Bl / 6J male mice (sourced from Jackson Laboratories), age 6-7 weeks (as in Example 2), were used in Study 5A, and Swiss:Rjorl male mice, age 5 weeks (sourced from Janvier), were used in Study 5B. [000215] For these studies (5A / 5B), mice were placed under isoflurane anaesthesia (5% for induction and 2% for maintenance, under 100% O2) and given 5 mg / kg s.c. carprofen (Rimadyl®). They were then placed in a stereotaxic frame. A midline sagittal incision was made in the scalp and a hole was drilled in the skull over the left lateral ventricle. A stainless-steel cannula (external diameter 0.51 mm) was placed stereotaxically into the left lateral ventricle at the following coordinates: +0.5 posterior to Bregma, L ± 0.7 mm, V = -2.7 mm. After a 2- minute delay to allow the brain tissue to slide over the cannula, 4 µL of a solution containing 0.625 mg / mL of RG4326 was slowly infused over 2 minutes. After infusion, the cannula was left in place for a further 5 minutes to prevent backflow of the solution along the cannula track. [000216] The mice were given 5 mg / kg s.c. carprofen (Rimadyl®) at 24 and 48 hours, after surgery. [000217] Mice were monitored during 3-7 days after surgery (starting 24 h after ICV administration) and their body weight was taken daily to check their health status. For mice monitored over 7 days, body weight was taken on Day 1 and on Day 7 after surgery to check their health status. [000218] RG4326 was evaluated at four doses in 4 separate groups as described in Table 13 below: Table 13: Design of MTD Study for RG4326 Group NumberTreatment Dose-levelConcentration Administration of animals (RG4326)(mg / mL) Volume 1 4 males RG4326 (i.c.v.) 3 mg / mouse 0.75 mg / mL 4 mL / mouse 2 4 males RG4326 (i.c.v.) 4 mg / mouse 1 mg / mL 4 mL / mouse 3 4 males RG4326 (i.c.v.) 5 mg / mouse 1.25 mg / mL 4 mL / mouse 4 4 males RG4326 (i.c.v.) 7.5 mg / mouse 1.875 mg / mL 4 mL / mouse [000219] Combined results from both Studies 5A and 5B demonstrated that RG4326 was generally well-tolerated in test subjects. In Study 5A, 6 mice were injected with 4 µL of a 41   Attorney Docket No.01138-0045-00PCT solution at 0.625 mg / mL (2.5 ug total per ICV). At the end of anesthesia, the mice remained lying on one side. They were quiet with some periods of scratching during the first hours after surgery. No toxic effects were observed at 24, 48 or 72 hours in the 6 mice administered. In Study 5B, four mice were injected with 4 different doses of RG4326 (0.75, 1.0, 1.25 and 1.875 mg / mL, volume of 4 µL). One mouse that received the highest dose (1.875 mg / mL, i.e., 7.5 µg / mouse) was found dead around 24 hours after ICV injection. All other mice were in good health, until the end of the pilot study (7 days after administration). Table 14 summarizes the MTD data for RG4326 for the Study 5A and 5B mice models. Based on these results, an MTD of 4 ug was selected for the 6-HZ studies of Examples 6 and 7. Table 14: 7-Day Survival Data for MTD Studies 5A and 5BExample 6 RG4326 In 6-Hz Seizure Model #1 [000220] Example 6 (and Example 7) involved study of RG4326 for anticonvulsant effects in the 6-Hz psychomotor seizure test in mice. The 6 Hz Psychomotor Test method used in these Examples followed that described by Brown et al. (J. Pharmacol. Exp. Ther.107, 273-283, 1953). Before transcorneal stimulation, a drop of tetracaine solution (1%) was applied on each eye of the mouse for local anesthesia. Between 1-10 minutes later, the mice were administered a rectangular current (44 mA, rectangular pulse: 0.2 ms pulse width, 3 s duration, (6 Hz) via corneal electrodes connected to a constant current shock generator (Ugo Basile: type 7801). 42   Attorney Docket No.01138-0045-00PCT [000221] In this example, six groups were used, with 15 mice in each group. Prior to 6-Hz induced seizure, as described in the immediately preceding paragraph, the mice were pre-treated with RG4326 at three dosages (24 hr before the test), with vehicle as a control, and with perampanel (60 min. before the test), as a reference. The design of this study is further described below in Table 15. Table 15: Study Design for 6-Hz Induced Seizure Experiment of Example 6 Group Number of Treatment Dose-level Concentration Administration animals (mg / mL) Volume215Perampanel (p.o.) 4 mg / kg 0.4 10 mL / kg315 PBS or scrambled anti-miRNA (i.c.v.) PBS 0 4 µL / mouse415RG4326 (ICV) 2.0 µg 0.5 mg / ml 4 µL / mouse515RG4326 (ICV) 0.5 µg 0.125 mg / ml 4 µL / mouse615RG4326 (ICV) 0.25 µg 0.0625 mg / ml 4 µL / mouse* “Control substance” was 0.2% HPMC in distilled water. [000222] Mice were placed under isoflurane anaesthesia (5% for induction and 2% for maintenance, under 100% O2) and given 5 mg / kg s.c. carprofen (Rimadyl®). They were then placed in a stereotaxic frame. A midline sagittal incision was made in the scalp and a hole was drilled in the skull over the left lateral ventricle. A unilateral cannula (Plastics One, Phymep) was placed stereotaxically into the left lateral ventricle using the following coordinates: +0.5 posterior to Bregma, L ±0.7 mm, V = -2.7 mm and was secured on the skull with dental cement. Following surgery, implanted mice were kept in individual macrolon cages and were allowed at least 5-7 days to recover. Mice were given 5 mg / kg s.c. carprofen (Rimadyl®) at 24 and 48 hours, after surgery. For this Example 6, a total of 64 animals was implanted over 2 days in order to obtain at least 15 mice per group. [000223] Around 5-7 days after surgery, RG4326 was administered by ICV to the mice in groups 4 to 6. The substances were injected ICV slowly between 2 and 4 minutes (1 to 2 µL / minute). 24 hours after the RG4326 was administered, the 6-Hz induced seizure test was conducted. For the group 2 mice (perampanel), the reference compound (4 mg / kg), was administered by mouth 60 minutes before the 6-Hz seizure test was conducted. 43   Attorney Docket No.01138-0045-00PCT [000224] The results for the number of seizures as reflected by forelimb clonus and / or Straub tail were recorded immediately after current administration. Seizure was recorded as absent (0 = no seizure) or present (1 = seizure – forelimb clonus, or Straub tail). [000225] The data from the study of this Example 6 is reported in Figures 8 and 9, which report the percentage of animals in each group exhibiting forelimb clonus (Figure 8) and Straub tail (Figure 9) seizures post the 6=Hz current administration. In neutral vehicle controls (0.2% HPMC in distilled water) administered p.o.60 minutes before the test, the mean forelimb seizure score was 0.9 ± 0.1. In vehicle controls (PBS) administered ICV 24 hours before the test, the mean forelimb seizure score was 13 / 15 mice, and 14 / 15 mice for Straub tail. RG4326 (0.0625, 0.125 and 0.5 mg / mL, i.e., 0.25, 0.5 and 2 µg / mouse), administered ICV 24 hours before the test, showed significant modification of seizure score at the top dose of 2.0 ug, as compared with vehicle controls (Fisher’s Exact test, p < 0.05). Perampanel (4 mg / kg), administered p.o.60 minutes before the test, fully suppressed forelimb clonus and Straub tail, as compared with neutral vehicle controls (p < 0.001). These results suggest anticonvulsant effects for RG4326 against 6-Hz, over the tested dose-range 0.0625-0.5 mg / mL (0.25, 0.5 and 2 µg / mouse) ICV, with 2.0 ug showing the strongest effect. Example 7 RG4326 In 6-Hz Model #2 [000226] The 6-HZ study of Example 7 was repeated with different dose and pre-treatment times for administration of the RG4326 and a different dose for perampanel. In this Example, the impact of RG4326 on 6-HZ induced CNS effects was evaluated with RG4326 being administered at 3-4 hr and at 24 hr before the 6-Hz administration (as opposed to only 24 hr as used in Example 6). In Example 7, perampanel (2 mg / kg p.o.), was administered p.o.60 minutes before the test at a dose of 2 mg / kg (compared to 4 mg / kg in Example 6). A total of 75 animals was implanted over 4 days to obtain at least 15 mice per group for this experiment. The design of this study is further described below in Table 16. Table 16: Study Design for 6-Hz Induced Seizure Experiment of Example 7 Group Number of Compound Route / Dose-level Concentration Administration animals Treatment (mg / mL) Volumescrambled anti- miRNA 5 15 RG4326 i.c.v., 3-4 hours 4 µg / mouse 1.0 4 µL / mouse before the test 44   Attorney Docket No.01138-0045-00PCT 15 RG4326 i.c.v., 3-4 hours 2.0 µg / mouse 0.5 4 µL / mouse before the test 15 RG4326 i.c.v., 3-4 hours 0.5 µg / mouse 0.125 4 µL / mouse before the test 15 RG4326 i.c.v., 24 hours 4 µg / mouse 1.0. 4 µL / mouse before the test 15 Control p.o., 1 hour 0 0 10 mL / kg Substance* before the test 0 15 Perampanel p.o., 1 hour 2 mg / kg 0.2 10 mL / kg before the test * “Control substance” was 0.2% HPMC in distilled water. [000227] Quantitative data (scores) with the test substance were analyzed by comparing treated groups with vehicle control using Fisher’s Exact test. The results of this study are reported in Figure 10 and below in Table 17. [000228] In the neutral vehicle control group (0.2% HPMC in distilled water administered p.o.60 minutes before the test), the mean forelimb seizure score was 0.9 ± 0.1 and all mice displayed Straub tail (15 / 15). In the vehicle control group (PBS administered ICV 3-4 hours before the test), the mean forelimb seizure score was 0.7 ± 0.2 and all mice displayed Straub tail (15 / 15). RG4326 (0.5 mg / mL, i.e., 2 µg / mouse), administered ICV 3-4 hours before the test, significantly decreased the number of mice showing Straub tail, as compared with vehicle controls (-33%, p < 0.05). Interestingly, at this dose RG4326 did not affect the forelimb seizure score (compared to data in Figure 8, showing 6-Hz after 24 hrs post-ICV). No effects were observed at 0.125 and 1 mg / mL, i.e., 0.5 and 4 µg / mouse on these parameters. In the group treated at 1 mg / mL, three mice showed marked sedation and decreased respiration. RG4326 (1 mg / mL, i.e., 4 µg / mouse), administered ICV 24 hours before the test, significantly decreased the number of mice showing Straub tail as compared with vehicle controls (-53%, p < 0.01). Perampanel (2 mg / kg), administered p.o.60 minutes before the test, significantly decreased the mean forelimb clonus as compared with neutral vehicle controls (-67%, p < 0.01), but compared to RG4326 had no effects on the number of mice showing Straub tail. [000229] Combined, the results show anticonvulsant effects with RG4326 at 0.5 mg / mL, administered ICV 3-4 hours before the test, in the 6 Hz Test in the mouse. Anticonvulsant effects were also seen with RG4326 at 1 mg / mL, administered ICV 24 hours before the test. It was noted that at 1 mg / mL RG4326, three mice showed marked sedation and decreased respiration. [000230] In summary, RG4326 was profiled in two studies involving the 6-Hz psychomotor test, in Examples 6 and 7 reported herein. In the Example 6 study, RG4326 was found to reduce seizure incidence (forelimb clonus and Straub tail), following 2.0 ug ICV at 24 45   Attorney Docket No.01138-0045-00PCT hrs prior to 6-Hz. In the Example 7 study, a higher dosing of RG4326 at 24 hrs prior to 6-Hz current was evaluated, as well as the impact of administering RG4326 ICV closer in time to administration of the 6-Hz current (i.e., 3-4 hrs). Results from the Example 7 study demonstrated improved seizure suppression for RG4326 at 4.0 ug, when administered -24 hrs (Straub tail), in addition to seizure suppression by RG4326 at 2.0 ug when administered 3-4 hrs before 6-Hz. Table 17: Post 6 Hz Current Straub Tail Seizure Data (Percentage) for Example 7 GroupsFisher’s Exact test: NS = Not Significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Example 8 Evaluation of AMPA-R compounds against glutamate and AMPA-induced excitotoxicity in rat primary cortical neurons [000231] The compounds shown in Table 18 were evaluated against glutamate and AMPA- induced excitotoxicity in rat primary cortical neurons. Table 18: Compounds Evaluated46   Attorney Docket No.01138-0045-00PCTTest System [000232] The rat primary cortical neurons were obtained from E18-19 rat embryos (Sprague-Dawley rats). Cell culture [000233] Cortices were harvested from E18-19 rat embryos and dissociated enzymatically and mechanically. Dissociated cells (10,000 cells / well) were plated in poly-D-lysine coated imaging plates (384 wells), in 70 µL of neuronal growth medium (Neurobasal + 2% SM1 neuronal supplements + L-glutamine + HEPES). Cells were incubated at 37°C, 5% CO2. [000234] Half of the medium was changed twice per week. A total of 5 plates were prepared for the experiment. Evaluation of Glutamate or AMPA-induced excitotoxicity [000235] After 9 days in vitro, half of the medium was removed and 35 µL of the test items were added at 2X concentrated in the culture medium. [000236] Twenty-four hours later, AMPA or glutamate were added to the wells and the wells were treated as follows: - wells incubated with test items: 17.5 µL was removed and 17.5 µL of 4X glutamate or AMPA was added. - wells incubated with reference substances: 35 µL was removed and 17.5 µL of 4X reference substance (MK-801, Perampanel or CNQX) was added to each well followed by 17.5 µL of 4X glutamate or AMPA. [000237] After 72 hours, half of the supernatant was discarded and an intracellular ATP content assay was performed using an ATP kit (CellTiter-Glo® 3D Cell Viability Assay). [000238] Each experimental condition was tested in 4 wells, as set forth below. Plate 1 Number of wells Treatment 1 Concentration Treatment 2 4 NT 0 0 4 PBS 0.1% Glutamate at 30 µM 4 PBS 0.2% Glutamate at 30 µM 4 DMSO 0.1% Glutamate at 30 µM 4 MK-801(*) 10 µM Glutamate at 30 µM 4 Perampanel (*) 0.1; 1; 3; 10, 30 µM Glutamate at 30 µM 4 RG8431 1, 5, 10, 25, 50 µM Glutamate at 30 µM 47   Attorney Docket No.01138-0045-00PCT 4 RG4326 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG4047 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8524 0.25, 1, 2.5, 5, 10 µM Glutamate at 30 µM 4 RG8525 0.25, 1, 2.5, 5, 10 µM Glutamate at 30 µM 4 RG6224 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8534 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8535 1, 5, 10, 25, 50 µM Glutamate at 30 µM (*) Treatment given in parallel with Glutamate Plate 2 Number of wells Treatment 1 Concentration Treatment 2 4 NT 0 0 4 PBS 0.1% AMPA at 100 µM 4 PBS 0.2% AMPA at 100 µM 4 DMSO 0.1% AMPA at 100 µM 4 CNQX (*) 25 µM AMPA at 100 µM 4 Perampanel (*) 0.1; 1; 3; 10, 30 µM AMPA at 100 µM 4 RG8431 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG4326 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG4047 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8524 0.25, 1, 2.5, 5, 10 µM AMPA at 100 µM 4 RG8525 0.25, 1, 2.5, 5, 10 µM AMPA at 100 µM 4 RG6224 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8534 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8535 1, 5, 10, 25, 50 µM AMPA at 100 µM (*) Treatment given in parallel with AMPA [000239] Glutamate significantly decreased ATP content, compared to Non-Treated (NT) control. See Fig.11A. MK-81 and Perampanel each significantly increased ATP content in the presence of glutamate. See Figs.11A and 11B. [000240] RG8431, RG4326, RG4047, RG6224, RG8534, and RG8535 significantly increased ATP content in the presence of glutamate. RG8525 showed a slight but significant increase in ATP content at 1 µM, and RG8524 showed no significant effects were observed for RG8524 at higher doses. See Figs.12A-12H. [000241] AMPA significantly decreased ATP content as compared to Non-Treated (NT) control. See Fig.13A. CNQX and Perampanel each significantly increased ATP content in the 48   Attorney Docket No.01138-0045-00PCT presence of glutamate. See Figs.13A and 13B. [000242] RG4326, RG4047, RG6224, RG8534, and RG8535 significantly increased ATP content in the presence of AMPA. RG8431 did not change ATP content, while RG8524 and RG8525 significantly increased ATP content in the presence of AMPA at higher doses. See Figs.14A-14H. Calcium measurement [000243] Pilot experiment to determine AMPA dose [000244] After 8 days in vitro, the growth medium was discarded and replaced by 30 µL of a calcium probe in a saline solution (containing 1.5 mM calcium) for 60 min at 37°C / 5% CO2. [000245] For calcium measurement, AMPA, Perampanel, and CNQX were prepared at 6X concentrated (6µL was added to 30 µL). The final vehicle concentration in all conditions was adjusted. Basal calcium levels were measured for 1 minute before automated addition of the compounds or controls while recording. Intracellular calcium signals were further recorded for 5 to 10 minutes. The sampling rate was around 1 point per second. [000246] Each experimental condition was tested in quadruplicate wells and shown below. Plate 3 Number of wells Treatment 1 Concentration Treatment 2 Concentration 4 NT 0 NT 0 4 DMSO 0.1% NT 0 4 NT 0 AMPA 3, 10, 30, 100, 300 µM 4 DMSO 0.1% AMPA 3, 10, 30, 100, 300 µM 4 Perampanel 1 µM AMPA 3, 10, 30, 100, 300 µM 4 Perampanel 3 µM AMPA 3, 10, 30, 100, 300 µM 4 Perampanel 10 µM AMPA 3, 10, 30, 100, 300 µM 4 Perampanel 30 µM AMPA 3, 10, 30, 100, 300 µM 4 CNQX 0.1 µM AMPA 3, 10, 30, 100, 300 µM 4 CNQX 1 µM AMPA 3, 10, 30, 100, 300 µM 4 CNQX 10 µM AMPA 3, 10, 30, 100, 300 µM 4 CNQX 25 µM AMPA 3, 10, 30, 100, 300 µM [000247] AMPA at all doses significantly and dose-dependently increased calcium release as compared with the non-treated conditions (Fig.15A), AMPA with 0.1% DMSO at all doses also significantly and dose-dependently increased calcium release as compared with 0.1% DMSO (Fig.15B). 49   Attorney Docket No.01138-0045-00PCT [000248] Parampanel (1, 3, 10, and 30 µM) almost completely suppressed calcium release in the presence of AMPA at 3, 10, 30, and 100 µM and dose dependently decreased calcium release in the presence of AMPA at 300 µM (Fig.15C-15G). [000249] CNQX (1, 10, 25 µM) significantly and dose-dependently decreased calcium release in the presence of AMPA at 3 and 10 µM (with no clear effects observed at 0.1 µM). CNQX (10 and 25 µM) significantly and dose-dependently decreased calcium release in the presence of AMPA at 30 and 100 µM. CNQX did not decrease calcium release in the presence of AMPA at 300 µM in this experiment. (Fig.15H-15L). [000250] At the end of the pilot study, it was decided to use a dose of AMPA at 100 µM to induce a clear calcium response. Perampanel at 10 µM and CNQX at 25 µM were chosen as reference substances for the main experiment. [000251] Main experiment [000252] After 9 days in vitro, half of the medium was removed and 35 µL of the test substances were added at 2X concentration in the culture medium. [000253] Twenty-four hours later, the growth medium was discarded and replaced by 30 µL of a calcium probe in a saline solution (containing 1.5 mM calcium) for 60 min at 37°C / 5% CO2. [000254] For calcium measurement, glutamate and MK-801 or AMPA and Perampanel / CNQX were prepared at 6X concentrated (6µL was added to 30 µL). The final vehicle concentration in all conditions was adjusted. Basal calcium levels were measured for 1 minute before automated addition of the compounds or controls while recording. Intracellular calcium signals were further recorded for 5 to 10 minutes. The sampling rate was around 1 point per second. [000255] Each experimental condition was tested in quadruplicate wells as set forth below. Plate 4 Number of wells Treatment 1 Concentration Treatment 2 4 NT 0 0 4 PBS 0.1% Glutamate at 30 µM 4 PBS 0.2% Glutamate at 30 µM 4 DMSO 0.1% Glutamate at 30 µM 4 MK-801(*) 10 µM Glutamate at 30 µM 50   Attorney Docket No.01138-0045-00PCT 4 RG8431 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG4326 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG4047 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8524 0.25, 1, 2.5, 5, 10 µM Glutamate at 30 µM 4 RG8525 0.25, 1, 2.5, 5, 10 µM Glutamate at 30 µM 4 RG6224 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8534 1, 5, 10, 25, 50 µM Glutamate at 30 µM 4 RG8535 1, 5, 10, 25, 50 µM Glutamate at 30 µM (*) Treatment given in parallel with Glutamate Plate 5 Number of wells Treatment 1 Concentration Treatment 2 4 NT 0 0 4 PBS 0.1% AMPA at 100 µM 4 PBS 0.2% AMPA at 100 µM 4 DMSO 0.1% AMPA at 100 µM 4 Perampanel (*) 10 µM AMPA at 100 µM 4 CNQX (*) 25 µM AMPA at 100 µM 4 RG8431 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG4326 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG4047 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8524 0.25, 1, 2.5, 5, 10 µM AMPA at 100 µM 4 RG8525 0.25, 1, 2.5, 5, 10 µM AMPA at 100 µM 4 RG6224 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8534 1, 5, 10, 25, 50 µM AMPA at 100 µM 4 RG8535 1, 5, 10, 25, 50 µM AMPA at 100 µM (*) Treatment given in parallel with AMPA [000256] As shown in Fig.16, glutamate increased calcium release as compared with the Non-Treated (NT) control, and MK-801 significantly reduced calcium release in the presence of glutamate. [000257] RG8431 (1 and 5 µM), RG4326 (1 and 50 µM), RG4047 (1, 5, and 50 µM), RG8524 (0.25 and 10 µM), RG 8525 (0.25 and 10 µM), RG6224 (10, 25, and 50 µM) slightly but significantly increased calcium release when incubated 24 hours before addition of glutamate. RG8534 (50 µM) significantly increased calcium release, and RG8535 did not significantly affect calcium release. See Figs.17A-17H. 51   Attorney Docket No.01138-0045-00PCT [000258] As shown in Fig.18, AMPA increased calcium release as compared with the Non-Treated (NT) control, and CNQX and Perampanel significantly reduced calcium release in the presence of AMPA. [000259] RG8431 (1, 5, 10, 25, and 50 µM), RG4047 (1, 5, 10, 25, and 50 µM), and RG8524 (5 µM) significantly decreased calcium release in the presence of AMPA. RG4326 (1, 5, 10, 25, and 50 µM) and RG8535 (1, 5, 10, 25, and 50 µM) dose-dependently and significantly decreased calcium release, while RG8525, RG6224, and RG8534 did not significantly change calcium release. See Figs.19A-19H. [000260] These results demonstrate the neuroprotective effects of most of the tested compounds, when incubated 24 hours before glutamate or AMPA. 52

Claims

Attorney Docket No.01138-0045-00PCT What is claimed is:

1. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically-effective amount of a modified oligonucleotide consisting of 4- 15 linked nucleosides, wherein the last four nucleosides at the 3’ end of the modified oligonucleotide have the nucleobase sequence UUUG, and wherein at least two of the last four nucleosides at the 3’ end of the modified oligonucleotide are bicyclic nucleosides.

2. The method of claim 1, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleosides of the modified oligonucleotide are bicyclic nucleosides.

3. The method of claim 1 or claim 2, wherein each bicyclic nucleoside is independently selected from S-cEt nucleoside, LNA nucleoside, and ENA nucleoside.

4. The method of claim 3, wherein each bicyclic nucleoside is S-cEt nucleoside.

5. The method of any one of the preceding claims, wherein at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

6. The method of claim 5, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

7. The method of claim 5 or claim 6, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

8. The method of any one of claims 1-3, wherein the modified oligonucleotide consists of 4-9, 4-10, 4-11, 4-12, 4-13, or 4-14 linked nucleosides.

9. The method of any one of the preceding claims, wherein the modified oligonucleotide consists of 4 linked nucleosides.

10. The method of claim 9, wherein the modified oligonucleotide is UFUMUSGSor USUSUSGS, wherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides.

11. The method of any one of claims 1-8, wherein the modified oligonucleotide consists of 9 linked nucleosides.

12. The method of any one of claims 1-8 and 11, wherein the modified oligonucleotide comprises the nucleobase sequence 5’-AGCACUUUG-3’, wherein each cytosine is independently selected from a non-methylated cytosine and a 5-methylcytosine 13. The method of claim 11 or claim 12, wherein the modified oligonucleotide has the following nucleoside pattern in the 5’ to 3’ orientation: NSNSNMNFNFNFNMNSNS 53   Attorney Docket No.01138-0045-00PCT wherein nucleosides followed by subscript “M” are 2’-O-methyl nucleosides, nucleosides followed by subscript “F” are 2’-fluoro nucleosides, nucleosides followed by subscript “S” are S-cEt nucleosides.

14. The method of any one of the preceding clams, wherein the modified oligonucleotide is selected from the modified oligonucleotides in Table 1.

15. The method of any one of the preceding claims, wherein a pharmaceutically acceptable salt of the modified oligonucleotide is administered to the subject.

16. The method of claim 15, wherein the pharmaceutically acceptable salt is a sodium salt.

17. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically effective amount of a modified oligonucleotide having the structure: 54   Attorney Docket No.01138-0045-00PCTacceptable salt thereof.

18. The method of claim 17, comprising administering to the subject a pharmaceutically acceptable salt of the modified oligonucleotide.

19. The method of claim 18, comprising administering to the subject a sodium salt of the modified oligonucleotide.

20. A method of treating a nervous system disorder in a subject, comprising administering to the subject a therapeutically-effective amount of a modified oligonucleotide having the structure: 55   Attorney Docket No.01138-0045-00PCT.

21. The method of any one of the preceding claims, wherein the modified oligonucleotide is formulated in a pharmaceutical composition for administration to the subject, wherein the pharmaceutical composition comprises the modified oligonucleotide in an aqueous solution.

22. The method of claim 21, wherein the aqueous solution is a saline solution.

23. The method of any one of the preceding claims wherein the nervous system disorder is hearing loss, motor disease, amyotrophic lateral sclerosis (ALS), pain, Parkinson's disease, neuroprotection in post-traumatic brain injury (TBI), stroke, or glioblastoma. 56   Attorney Docket No.01138-0045-00PCT 24. The method of any one of the preceding claims, wherein the nervous system disorder is epilepsy.

25. The method of any one of the preceding claims, comprising administering to the subject at least one additional therapy.

26. The method of claim 25, wherein the additional therapy is an anti-epileptic drug. 57

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