Methods and compositions for treating epilepsy

Abstract

Disclosed are methods and compositions relating to antisense therapy for treating epilepsy, such as temporal lobe epilepsy, in a subject in need thereof by targeting Grik2 mRNA. In particular, the disclosure provides inhibitory RNA constructs capable of inhibiting expression of GluK2 protein and inhibiting epileptiform discharges in hyperexcitable neural circuits.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on April 28, 2022 is titled "51460-007WO3_Sequence_Listing_4_28_22_ST25" and is 297,127 bytes in size.

[0002] Field of Disclosure The present disclosure is in the field of epilepsy. In particular, the present disclosure relates to methods and compositions for treating epilepsy, such as temporal lobe epilepsy. [Background technology]

[0003] background Globally, an estimated 5 million people are diagnosed with epilepsy each year, a neurological disorder characterized by seizures, or sudden, recurrent episodes of sensory disturbances, loss of consciousness, or convulsions related to abnormal electrical activity in the brain. A typical diagnosis of epilepsy occurs when a patient experiences two or more unprovoked seizures. Causes of epilepsy include genetic abnormalities, previous brain infections, prenatal injuries, developmental disorders, and other neurological problems such as stroke or brain tumors, although approximately 50% of people diagnosed with epilepsy have no known cause for the onset of the disorder.

[0004] Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy in adults (30–40% of all forms of epilepsy). It is well established that the hippocampus plays a key role in the pathophysiology of TLE. Abnormal reorganization of neuronal circuits occurs in human patients and animal models of TLE. One of the best examples of network reorganization ("response plasticity") is the sprouting of recurrent mossy fibers (rMFs), which establish novel pathophysiological glutamatergic synapses onto dentate gyrus granule cells (DGCs) in the hippocampus (Tauck and Nadler, 1985; Represa et al., 1989a, 1989b; Sutula et al., 1989; Gabriel et al., 2004), resulting in a recurrent excitatory loop. rMF synapses operate via ectopic kainate receptors (KARs) (Epsztein et al., 2005; Artinian et al., 2011, 2015). KARs are tetrameric glutamate receptors assembled from GluK1-GluK5 subunits. In heterologous expression systems, GluK1, GluK2, and GluK3 can form homomeric receptors, whereas GluK4 and GluK5 form heteromeric receptors with GluK1-3 subunits. Native KARs are widely distributed in the brain, with high densities found in the hippocampus, a key structure involved in TLE (Carta et al., 2016, EJN). Previous studies by the present inventors established that epileptic activity, including interictal spikes and paroxysmal discharges, is significantly reduced in mice lacking the GluK2 KAR subunit. Furthermore, epileptiform activity was strongly reduced after the use of a pharmacological small molecule antagonist of GluK2 / GluK5-containing KARs, which blocks ectopic synaptic KARs (Peret et al., 2014). These data support the hypothesis that ectopically expressed KARs in rMFs in the DGC play a key role in chronic seizures in TLE. Therefore, abnormal KARs expressed in the DGC and composed of GluK2 / GluK5 may be a promising target for the treatment of drug-resistant epilepsy, such as TLE. RNA interference (RNAi) strategies have been proposed for many disease targets. The application of RNAi-based therapies has had limited success. RNAi therapeutics face multiple challenges, such as the need for repeated administration and formulation issues. However, only limited RNAi-based gene therapy is available for the treatment of refractory TLE. Therefore, new therapeutic modalities are urgently needed for the treatment of seizure disorders, such as TLE (e.g., treatment-refractory TLE). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tauck DL, Nadler JV (1985) Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats. J Neurosci 5:1016-1022 [Non-patent document 2] Represa A, Le Gall La Salle G, Ben-Ari Y (1989a) Hippocampal plasticity in the kindling model of epilepsy in rats. Neurosci Lett 99:345-350. [Non-patent document 3] Represa A, Robain O, Tremblay E, Ben-Ari Y (1989b) Hippocampal plasticity in childhood epilepsy. Neurosci Lett 99:351-355. [Non-patent document 4] Sutula T, Cascino G, Cavazos J, Parada I, Ramirez L (1989) Mossy fiber synaptic reorganization in the epileptic human temporal lobe. Ann Neurol 26:321-330.

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0006] Summary of the Disclosure The present disclosure provides compositions and methods for treating or preventing epilepsy, such as temporal lobe epilepsy (TLE), in a subject (e.g., a human) in need thereof. The disclosed methods involve administering a therapeutically effective amount of a polynucleotide (e.g., an inhibitory polynucleotide), such as an antisense oligonucleotide (ASO), shRNA, siRNA, microRNA, or shmiRNA targeting mRNA encoded by the glutamate ionotropic receptor kainate subunit 2 (Grik2) gene, or a nucleic acid vector encoding the same (e.g., a lentiviral vector or an adeno-associated viral (AAV) vector, e.g., an AAV9 vector), to a subject diagnosed with or at risk of developing epilepsy. The disclosed polynucleotides exhibit improved loading into RNA-induced silencing complex (RISC) proteins to enhance RNA interference-mediated degradation of Grik2 transcripts. The present disclosure also features pharmaceutical compositions containing one or more of the disclosed inhibitory nucleic acid (e.g., RNA) agents and nucleic acid vectors encoding them.

[0007] The present disclosure is based, in part, on the surprising discovery that the inhibitory polynucleotides described herein exhibit significantly higher guide-to-passenger strand ratios (G / P ratios), which supports a direct and substantial increase in inhibitory polynucleotide processing and subsequent improved reduction in expression levels of both Grik2 mRNA and the resulting GluK2 protein. A challenge with microRNA (miRNA) therapeutics is the low processing efficiency of transfected polynucleotides. Thus, an improved G / P ratio may correlate with increased production of mature miRNA molecules and, concomitantly, an increase in the desired therapeutic effect(s) of the administered miRNA therapy.

[0008] In a first aspect, the disclosure features an isolated inhibitory polynucleotide(s) that specifically hybridizes to Grik2 mRNA, the polynucleotide(s) comprising a stem-loop region including a 5' arm (5p), a loop region, and a 3' arm (3p), the stem-loop region comprising a guide strand sequence and a passenger strand sequence, the guide strand sequence and the passenger strand sequence comprising: (a) a uracil (U)-adenine (A) base pair or a U-guanine (G) base pair at the 5' end of the guide strand, (b) a cytosine (C)-G pair at the 5' end of the passenger strand, (c) a U at the 5' end of the guide strand sequence, (d) a mismatch in the seed region between the guide strand sequence and the passenger strand sequence; and / or (e) a CG base pair or a UA base pair to replace a UG wobble at the junction of the stem and loop regions of the polynucleotide.

[0009] In some embodiments, a) and c) improve loading of the guide strand sequence into an RNA-induced silencing complex (RISC) protein. In some embodiments, b) impairs loading of the passenger strand sequence into a RISC protein. In some embodiments, d) promotes decoupling of the passenger strand sequence from the guide strand sequence during RISC loading. In some embodiments, e) improves cleavage of the loop region from the stem region by Dicer. In some embodiments, the seed region of the guide strand sequence comprises nucleotides 2-7 of the guide strand sequence.

[0010] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:17. In some embodiments, the guide strand of SEQ ID NO:17 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:17 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:32. In some embodiments, the passenger strand of SEQ ID NO: 32 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 32 shown in Table 3.

[0011] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the guide strand of SEQ ID NO: 18 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 18 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the passenger strand of SEQ ID NO: 33 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 33 shown in Table 3.

[0012] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:19. In some embodiments, the guide strand of SEQ ID NO:19 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:19 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the passenger strand of SEQ ID NO: 34 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 34 shown in Table 3.

[0013] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the guide strand of SEQ ID NO:20 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:20 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:35. In some embodiments, the passenger strand of SEQ ID NO: 35 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 35 shown in Table 3.

[0014] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:21. In some embodiments, the guide strand of SEQ ID NO:21 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:21 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:36. In some embodiments, the passenger strand of SEQ ID NO: 36 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 36 shown in Table 3.

[0015] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:22. In some embodiments, the guide strand of SEQ ID NO:22 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:22 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:37. In some embodiments, the passenger strand of SEQ ID NO: 37 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), and the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 37 shown in Table 3.

[0016] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:23. In some embodiments, the guide strand of SEQ ID NO:23 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:23 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:38. In some embodiments, the passenger strand of SEQ ID NO: 38 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 38 shown in Table 3.

[0017] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:24. In some embodiments, the guide strand of SEQ ID NO:23 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:23 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:39. In some embodiments, the passenger strand of SEQ ID NO: 39 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 39 shown in Table 3.

[0018] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the guide strand of SEQ ID NO: 25 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 25 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the passenger strand of SEQ ID NO: 40 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 40 shown in Table 3.

[0019] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the guide strand of SEQ ID NO: 4 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 26 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the passenger strand of SEQ ID NO:41 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:41 shown in Table 3.

[0020] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the guide strand of SEQ ID NO: 27 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 27 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the passenger strand of SEQ ID NO:42 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:42 shown in Table 3.

[0021] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the guide strand of SEQ ID NO: 28 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 28 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 43. In some embodiments, the passenger strand of SEQ ID NO:43 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:43 shown in Table 3.

[0022] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the guide strand of SEQ ID NO: 29 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 29 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the passenger strand of SEQ ID NO:44 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:44 shown in Table 3.

[0023] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the guide strand of SEQ ID NO: 30 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 30 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the passenger strand of SEQ ID NO:45 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:45 shown in Table 3.

[0024] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 226. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 230. In some embodiments, the guide strand of SEQ ID NO: 230 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 230 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 234. In some embodiments, the passenger strand of SEQ ID NO:234 ​​contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:234 ​​shown in Table 3.

[0025] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 227. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 231. In some embodiments, the guide strand of SEQ ID NO: 231 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 231 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 235. In some embodiments, the passenger strand of SEQ ID NO: 235 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 235 shown in Table 3.

[0026] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 228. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 232. In some embodiments, the guide strand of SEQ ID NO: 232 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 232 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 236. In some embodiments, the passenger strand of SEQ ID NO: 236 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 236 shown in Table 3.

[0027] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 229. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 233. In some embodiments, the guide strand of SEQ ID NO: 233 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 233 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 237. In some embodiments, the passenger strand of SEQ ID NO: 237 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 237 shown in Table 3.

[0028] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 238. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 242. In some embodiments, the guide strand of SEQ ID NO: 242 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 242 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 246. In some embodiments, the passenger strand of SEQ ID NO:246 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:246 shown in Table 3.

[0029] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 239. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 243. In some embodiments, the guide strand of SEQ ID NO: 243 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 243 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 247. In some embodiments, the passenger strand of SEQ ID NO:247 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:247 shown in Table 3.

[0030] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 240. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 244. In some embodiments, the guide strand of SEQ ID NO: 244 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 244 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 248. In some embodiments, the passenger strand of SEQ ID NO:248 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:248 shown in Table 3.

[0031] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 241. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 245. In some embodiments, the guide strand of SEQ ID NO: 245 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 245 shown in Table 3. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 249. In some embodiments, the passenger strand of SEQ ID NO: 249 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 249 shown in Table 3.

[0032] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:47. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:64. In some embodiments, the guide strand of SEQ ID NO:64 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:64 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:81. In some embodiments, the passenger strand of SEQ ID NO: 81 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 81 shown in Table 5.

[0033] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:48. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:65. In some embodiments, the guide strand of SEQ ID NO:65 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:65 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:82. In some embodiments, the passenger strand of SEQ ID NO: 82 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 82 shown in Table 5.

[0034] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:49. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:66. In some embodiments, the guide strand of SEQ ID NO:66 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:66 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:83. In some embodiments, the passenger strand of SEQ ID NO: 83 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 83 shown in Table 5.

[0035] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO:50. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:67. In some embodiments, the guide strand of SEQ ID NO:67 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO:67 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:84. In some embodiments, the passenger strand of SEQ ID NO: 84 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 84 shown in Table 5.

[0036] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the guide strand of SEQ ID NO: 68 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 68 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 85. In some embodiments, the passenger strand of SEQ ID NO: 85 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 85 shown in Table 5.

[0037] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 69. In some embodiments, the guide strand of SEQ ID NO: 69 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 69 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the passenger strand of SEQ ID NO: 86 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 86 shown in Table 5.

[0038] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 70. In some embodiments, the guide strand of SEQ ID NO: 70 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 70 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 87. In some embodiments, the passenger strand of SEQ ID NO: 87 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), and the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 87 shown in Table 5.

[0039] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the guide strand of SEQ ID NO: 71 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 71 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 88. In some embodiments, the passenger strand of SEQ ID NO: 88 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 88 shown in Table 5.

[0040] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 72. In some embodiments, the guide strand of SEQ ID NO: 72 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 72 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 89. In some embodiments, the passenger strand of SEQ ID NO: 89 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 89 shown in Table 5.

[0041] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 73. In some embodiments, the guide strand of SEQ ID NO: 73 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 73 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 90. In some embodiments, the passenger strand of SEQ ID NO: 90 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 90 shown in Table 5.

[0042] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 74. In some embodiments, the guide strand of SEQ ID NO: 74 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 74 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 91. In some embodiments, the passenger strand of SEQ ID NO: 91 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 91 shown in Table 5.

[0043] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 58. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, the guide strand of SEQ ID NO: 75 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 75 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 92. In some embodiments, the passenger strand of SEQ ID NO: 92 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 92 shown in Table 5.

[0044] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the guide strand of SEQ ID NO: 76 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 76 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 93. In some embodiments, the passenger strand of SEQ ID NO: 93 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 93 shown in Table 5.

[0045] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, the guide strand of SEQ ID NO: 77 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 77 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, the passenger strand of SEQ ID NO: 94 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 94 shown in Table 5.

[0046] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 78. In some embodiments, the guide strand of SEQ ID NO: 78 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 78 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 95. In some embodiments, the passenger strand of SEQ ID NO: 95 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 95 shown in Table 5.

[0047] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 62. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, the guide strand of SEQ ID NO: 79 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 79 shown in Table 5. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 96. In some embodiments, the passenger strand of SEQ ID NO: 96 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 96 shown in Table 5.

[0048] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 98. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 110. In some embodiments, the guide strand of SEQ ID NO: 110 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 110 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the passenger strand of SEQ ID NO: 122 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 122 shown in Table 7.

[0049] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 111. In some embodiments, the guide strand of SEQ ID NO: 111 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 111 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 123. In some embodiments, the passenger strand of SEQ ID NO: 123 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 123 shown in Table 7.

[0050] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 100. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 112. In some embodiments, the guide strand of SEQ ID NO: 112 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 112 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 124. In some embodiments, the passenger strand of SEQ ID NO: 124 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 124 shown in Table 7.

[0051] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 113. In some embodiments, the guide strand of SEQ ID NO: 113 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 113 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the passenger strand of SEQ ID NO: 125 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 125 shown in Table 7.

[0052] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 102. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 114. In some embodiments, the guide strand of SEQ ID NO: 114 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 114 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 126. In some embodiments, the passenger strand of SEQ ID NO: 126 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 126 shown in Table 7.

[0053] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 103. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 115. In some embodiments, the guide strand of SEQ ID NO: 115 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 115 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 127. In some embodiments, the passenger strand of SEQ ID NO: 127 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 127 shown in Table 7.

[0054] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 104. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 116. In some embodiments, the guide strand of SEQ ID NO: 116 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 116 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 128. In some embodiments, the passenger strand of SEQ ID NO: 128 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 128 shown in Table 7.

[0055] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 105. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 117. In some embodiments, the guide strand of SEQ ID NO: 117 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 117 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 129. In some embodiments, the passenger strand of SEQ ID NO: 129 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 129 shown in Table 7.

[0056] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 106. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 117. In some embodiments, the guide strand of SEQ ID NO: 118 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 118 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 130. In some embodiments, the passenger strand of SEQ ID NO: 130 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 130 shown in Table 7.

[0057] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 107. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 119. In some embodiments, the guide strand of SEQ ID NO: 119 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 119 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 131. In some embodiments, the passenger strand of SEQ ID NO: 131 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 131 shown in Table 7.

[0058] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 108. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 120. In some embodiments, the guide strand of SEQ ID NO: 120 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 120 shown in Table 7. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 132. In some embodiments, the passenger strand of SEQ ID NO: 132 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 132 shown in Table 7.

[0059] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 140. In some embodiments, the guide strand of SEQ ID NO: 140 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 140 shown in Table 9. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 146. In some embodiments, the passenger strand of SEQ ID NO: 146 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 146 shown in Table 9.

[0060] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 135. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 141. In some embodiments, the guide strand of SEQ ID NO: 141 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 141 shown in Table 9. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the passenger strand of SEQ ID NO: 147 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 147 shown in Table 9.

[0061] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 136. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 142. In some embodiments, the guide strand of SEQ ID NO: 142 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 142 shown in Table 9. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 148. In some embodiments, the passenger strand of SEQ ID NO: 148 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 148 shown in Table 9.

[0062] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 137. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 143. In some embodiments, the guide strand of SEQ ID NO: 143 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 143 shown in Table 9. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 149. In some embodiments, the passenger strand of SEQ ID NO: 149 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 149 shown in Table 9.

[0063] In some embodiments, the stem-loop region is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 138. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 144. In some embodiments, the guide strand of SEQ ID NO: 144 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 144 shown in Table 9. In some embodiments, the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 150. In some embodiments, the passenger strand of SEQ ID NO: 150 contains 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatches), wherein the change(s) do not include any of the bolded nucleotides of SEQ ID NO: 150 shown in Table 9.

[0064] In some embodiments, the inhibitory polynucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the inhibitory polynucleotide comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a short hairpin-compatible miRNA (shmiRNA).

[0065] In some embodiments, the polynucleotide is 19 to 21 nucleotides. In some embodiments, the polynucleotide is 19 nucleotides. In some embodiments, the polynucleotide is 20 nucleotides. In some embodiments, the polynucleotide is 21 nucleotides.

[0066] In some embodiments, Grik2 mRNA is encoded by a nucleic acid sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to any one of SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174. In some embodiments, Grik2 mRNA is encoded by the nucleic acid sequence of SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, or SEQ ID NO: 174.

[0067] In some embodiments, the inhibitory polynucleotide is capable of reducing the level of GluK2 protein in a cell (as discussed further in this disclosure). In some embodiments, the polynucleotide reduces the level of GluK2 protein in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some embodiments, the cell is a neuron, such as a hippocampal neuron (e.g., a dentate gyrus granule cell (DGC) or a glutamatergic pyramidal neuron). In other embodiments, including those in which the cell is a neuron, the cell is a human cell.

[0068] In another aspect, the disclosure features a vector including a polynucleotide of any one of the preceding aspects and embodiments. In some embodiments, the vector is replication-deficient. In some embodiments, the vector is a mammalian, insect, bacterial, or viral vector. In some embodiments, the vector is an expression vector. In some embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), retrovirus, adenovirus, parvovirus, coronavirus, negative-strand RNA virus, orthomyxovirus, rhabdovirus, paramyxovirus, positive-strand RNA virus, picornavirus, alphavirus, double-stranded DNA virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox virus, and canarypox virus. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is an AAV5, AAV9, or AAVrhlO vector.

[0069] In another aspect, the disclosure features an expression cassette including a polynucleotide that encodes or includes a stem-loop region having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to a polynucleotide corresponding to a stem-loop sequence of the first aspect of the disclosure, e.g., a nucleic acid sequence of any one of SEQ ID NOs: 1-15, 226-229, and 238-241. In some embodiments, the stem-loop region has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the stem-loop region has the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:135. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 258. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 259. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 260.In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 261. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the expression cassette comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 257.

[0070] In another aspect, the present disclosure provides an expression cassette comprising a polynucleotide comprising a stem-loop sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 46-62.

[0071] In another aspect, the present disclosure provides an expression cassette comprising a polynucleotide comprising a stem-loop sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 97-108.

[0072] In another aspect, the disclosure provides an expression cassette comprising a polynucleotide comprising a stem-loop sequence having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of any one of SEQ ID NOs: 133-138. In some embodiments, the stem-loop sequence has at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 135. In some embodiments, the stem-loop sequence has the nucleic acid sequence of SEQ ID NO: 135.

[0073] In some embodiments, the expression cassette comprises a 5' flanking region, a loop region, and a 3' flanking region. In some embodiments, the 5' flanking region comprises a polynucleotide having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of any one of SEQ ID NOs: 217, 220, or 223. In some embodiments, the 3' flanking region comprises a polynucleotide having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of any one of SEQ ID NOs: 218, 221, or 224. In some embodiments, the 5' flanking region comprises a 5' spacer sequence and a 5' flanking sequence. In some embodiments, the 3' flanking region comprises a 3' spacer sequence and a 3' flanking sequence. In some embodiments, the loop region comprises a microRNA loop sequence that is an E-miR-30, miR-218-1, or E-miR-124-3 sequence. In some embodiments, the microRNA loop sequence comprises a polynucleotide having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of any one of SEQ ID NOs: 219, 222, or 225. In some embodiments, the microRNA loop sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 222. In some embodiments, the microRNA loop sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO: 222.

[0074] In some embodiments, the expression cassette comprises a synapsin (hSyn) promoter or a calcium / calmodulin-dependent protein kinase II (CaMKII) promoter. In some embodiments, the expression cassette comprises a constitutive promoter containing a cytomegalovirus enhancer (e.g., CAG or CBA), a U6, H1, or 7SK promoter.

[0075] In another aspect, the disclosure provides an expression cassette comprising, from 5' to 3', (a) a first promoter sequence; (b) a stem having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241. (c) optionally, a second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241. In some embodiments, the expression cassette comprises, from 5' to 3', (a) a first promoter sequence; (b) a polynucleotide comprising a stem-loop sequence having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO:4; (c) optionally, a second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO:135. In some embodiments, the expression cassette comprises, from 5' to 3', (a) a first promoter sequence; (b) a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of SEQ ID NO: 4; (c) optionally, a second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of SEQ ID NO: 135.In some embodiments, the expression cassette comprises a sequence having at least 85% sequence identity to SEQ ID NO: 258 (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)).

[0076] In some embodiments, a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity thereto, comprises a passenger sequence that is complementary or substantially complementary to the guide sequence, and the passenger sequence is located 5' or 3' to the guide sequence. In some embodiments, a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity thereto, comprises a 5' flanking region located 5' to the guide sequence. In some embodiments, a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity thereto, comprises a 3' flanking region located 3' to the guide sequence.In some embodiments, a polynucleotide comprising a stem-loop sequence having the nucleic acid sequence of any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229, or 238-241, or a variant thereof having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity thereto, comprises a loop region located between the guide sequence and the passenger sequence, and the loop region comprises a microRNA loop sequence.

[0077] In some embodiments, the first promoter and / or the optional second promoter is selected from the group consisting of an hSyn promoter or a CaMKII promoter. The first and / or second promoter may also be selected from constitutive promoters containing a cytomegalovirus enhancer (e.g., CAG or CBA), U6, H1, and 7SK promoters.

[0078] In some embodiments, the 5' flanking region comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 217, 220, or 223. In some embodiments, the 3' flanking region comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 218, 221, or 224. In some embodiments, the microRNA loop sequence is an E-miR-30, miR-218-1, or E-miR-124-3 sequence. In some embodiments, the microRNA loop sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 219, 222, or 225. In some embodiments, the microRNA loop sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 222. In some embodiments, the microRNA loop sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO: 222.

[0079] In some embodiments, the expression cassette comprises a 5' inverted terminal repeat (ITR) sequence at the 5' end of the expression cassette and a 3'-ITR sequence at the 3' end of the expression cassette. In some embodiments, the 5'-ITR and 3'-ITR sequences are AAV2 5'-ITR and 3'-ITR sequences. In some embodiments, the 5'-ITR sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:208 or SEQ ID NO:209. In some embodiments, the 5'-ITR sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:208. In some embodiments, the 3'-ITR sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:210, SEQ ID NO:211, or SEQ ID NO:212. In some embodiments, the 3'-ITR sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:210, SEQ ID NO:211, or SEQ ID NO:212.

[0080] In some embodiments, the expression cassette further comprises an enhancer sequence. In one embodiment, an enhancer sequence may be located in an expression cassette or vector disclosed herein to enhance activity of a promoter in the expression cassette or vector (e.g., an enhancer sequence may be located 5' to a promoter sequence in an expression cassette or vector described herein). In some embodiments, the enhancer sequence comprises a polynucleotide having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO:207. In some embodiments, the enhancer sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:207.

[0081] In some embodiments, the expression cassette further comprises an intron sequence. In one embodiment, an intron sequence can be placed within an expression cassette or vector to improve expression of an inhibitory polynucleotide (e.g., an ASO (e.g., an miRNA sequence; e.g., an intron can be placed between the promoter and the nucleic acid sequence of an inhibitory polynucleotide). In some embodiments, the intron sequence is located between two or more inhibitory polynucleotide sequences (e.g., two or more miRNA sequences) described herein. In some embodiments, the intron sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:205 or SEQ ID NO:206. In some embodiments, the intron sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:205 or SEQ ID NO:206.

[0082] In some embodiments, the expression cassette further comprises one or more (e.g., 2, 3, 4, or 5) polyadenylation signal sequences (e.g., to improve nuclear export, translation, and stability of the inhibitory polynucleotide of an expression cassette or expression vector disclosed herein). The polyadenylation signal sequence can be located 3' of the terminal inhibitor polynucleotide sequence (e.g., an ASO sequence (e.g., miRNA sequence, etc.) disclosed herein) and / or 5' of the 3' ITR sequence. In some embodiments, the polyadenylation signal sequence is a rabbit beta-globin (RBG) polyadenylation signal. In some embodiments, the RBG polyadenylation signal comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. In some embodiments, the RBG polyadenylation signal comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215. In some embodiments, the polyadenylation signal sequence is a bovine growth hormone (BGH) polyadenylation signal sequence. In some embodiments, the BGH polyadenylation signal sequence comprises a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 216. In some embodiments, the BGH polyadenylation signal sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO: 216.

[0083] In some embodiments, the expression cassette further comprises one or more (e.g., 2, 3, 4, or 5) stuffer sequences. In some embodiments, the one or more (e.g., 2, 3, 4, or 5) stuffer sequences are disposed at the 3' end of the expression cassette (e.g., between the polyadenylation sequence and the 3' ITR sequence). In some embodiments, the one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:250. In some embodiments, the one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 90% (e.g., at least 91%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:250. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 90% (e.g., at least 91%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 251.In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, one or more (e.g., 2, 3, 4, or 5) stuffer sequences have the nucleic acid sequence of SEQ ID NO: 251.

[0084] In some embodiments, the expression cassette of any of the foregoing aspects and embodiments comprises, from 5' to 3', the following: (a) a 5' ITR sequence; (b) optionally, an enhancer sequence; (c) a first promoter sequence; (d) optionally, an intron sequence; (e) a polynucleotide comprising a stem-loop sequence; (f) optionally, a second promoter sequence; (g) optionally, a polynucleotide comprising a stem-loop sequence; (h) a polyadenylation signal sequence, for example, an RBG polyadenylation signal sequence; (i) one or more stuffer sequences; and (j) a 3' ITR.

[0085] In some embodiments, the expression cassette of the aforementioned aspects and embodiments comprises at least 70% (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the sequence of any one of SEQ ID NOs: 252-261. In some embodiments, the expression cassette of the aforementioned aspects and embodiments comprises at least 70% (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the sequence of any one of SEQ ID NOs: 256 and 258-261. In some embodiments, the expression cassette of the foregoing aspects and embodiments comprises at least 70% (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the sequence of SEQ ID NO: 261. In some embodiments, the expression cassette has the nucleic acid sequence of SEQ ID NO: 256.

[0086] In some embodiments, the expression cassette of the foregoing aspects and embodiments is incorporated into a vector of the foregoing aspects and embodiments. In some embodiments, the vector is a replication-deficient vector. In some embodiments, the vector is a mammalian, insect, bacterial, or viral vector. In some embodiments, the vector is an expression vector. In some embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), retrovirus, adenovirus, parvovirus, coronavirus, negative-strand RNA virus, orthomyxovirus, rhabdovirus, paramyxovirus, positive-strand RNA virus, picornavirus, alphavirus, double-stranded DNA virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox virus, and canarypox virus. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is an AAV5, AAV9, or AAVrhlO vector.

[0087] In another aspect, the present disclosure provides a method of inhibiting Grik2 expression in a cell, the method comprising contacting the cell with at least one polynucleotide of the preceding aspects and embodiments, the vector of the preceding aspects and embodiments, or the expression cassette of the preceding aspects and embodiments.

[0088] In some embodiments, the polynucleotide specifically hybridizes to Grik2 mRNA and inhibits or reduces expression of Grik2 in the cell (as discussed further in this disclosure). In some embodiments, the method reduces the level of Grik2 in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some embodiments, the method reduces the level of GluK2 protein in the cell. In some embodiments, the method reduces the level of GluK2 protein in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0089] In some embodiments, the cell is a human cell. In some embodiments, the cell is a neuron (e.g., a human neuron). In some embodiments, the neuron is a hippocampal neuron (e.g., a human hippocampal neuron). In some embodiments, the hippocampal neuron is a DGC (e.g., a human DGC or a pyramidal neuron). In some embodiments, the DGC comprises recurrent mossy fiber axons. The cell can also be a neuronal cell derived from an induced pluripotent stem cell (iPSC), such as an iPSC-derived glutamatergic neuron expressing Grik2.

[0090] In another aspect, the present disclosure provides a method of treating or ameliorating a disorder in a subject in need thereof, the method comprising administering to the subject at least one polynucleotide of the preceding aspects and embodiments, vector of the preceding aspects and embodiments, or expression cassette of the preceding aspects and embodiments.

[0091] In some embodiments, the disorder is epilepsy. In some embodiments, the epilepsy is temporal lobe epilepsy (TLE), chronic epilepsy, and / or intractable epilepsy. In some embodiments, the epilepsy is TLE. In some embodiments, the TLE is lateral TLE (lTLE), such as unilateral TLE and / or bilateral TLE. In some embodiments, the TLE is medial TLE (mTLE).

[0092] In some embodiments, the subject is a human.

[0093] In another aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleotide of the foregoing aspects and embodiments, a vector of the foregoing aspects and embodiments, or an expression cassette of the foregoing aspects and embodiments, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0094] In another aspect, the present disclosure provides a kit comprising the pharmaceutical composition of the foregoing aspects and a package insert, in some embodiments, the package insert comprises instructions for using the pharmaceutical composition in the methods of the foregoing aspects and embodiments.

[0095] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed technology. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0096] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," and (iii) the terms "including" and "comprising" may be understood to encompass the listed element or step, whether presented by itself or together with one or more additional elements or steps.

[0097] The term "about" refers to an amount that is ±10% of the recited value, and can be ±5% of the recited value or ±2% of the recited value.

[0098] The terms "3' untranslated region" and "3' UTR" refer to the region 3' to the stop codon of an mRNA molecule (e.g., Grik2 mRNA). The 3' UTR is not translated into protein but contains regulatory sequences important for polyadenylation, localization, stabilization, and / or translation efficiency of the mRNA transcript. Regulatory sequences in the 3' UTR can include enhancers, silencers, AU-rich elements, polyA tails, terminators, and microRNA recognition sequences. The terms "3' untranslated region" and "3' UTR" can also refer to the corresponding region of a gene encoding an mRNA molecule.

[0099] The terms "5' untranslated region" and "5' UTR" refer to the region of an mRNA molecule (e.g., Grik2 mRNA) that is 5' to the start codon. This region is important for regulating translation initiation. The 5' UTR can be completely untranslated, or in some organisms, a portion of the region can be translated. The transcription start site marks the beginning of the 5' UTR and ends one nucleotide before the start codon. In eukaryotes, the 5' UTR contains a Kozak consensus sequence that includes the start codon. The 5' UTR may contain cis-acting regulatory elements, also known as upstream open reading frames, that are important for regulating translation. This region may also have an upstream AUG codon and a stop codon. Given its high GC content, the 5' UTR may form secondary structures, such as hairpin loops, that play a role in regulating translation. The term "administration" refers to providing or giving a therapeutic agent (e.g., an inhibitory polynucleotide or a vector encoding the same that binds to and inhibits expression of Grik2 mRNA, as disclosed herein) to a subject by any effective route. Exemplary routes of administration are described herein and below (eg, intraventricular injection, intrathecal injection, intraparenchymal injection, intravenous injection, and stereotactic injection).

[0100] The term "AAV vector" or "adeno-associated viral vector" refers to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC16, AAV.HSC27, AAV.HSC38, AAV.HSC49, AAV.HSC51, AAV.HSC52, AAV.HSC53, AAV.HSC54 An AAV vector, which refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16, can have one or more AAV wild-type genes deleted in whole or in part, such as the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences facilitate rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein as containing at least the sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type polynucleotide sequences, but can be altered, for example, by the insertion, deletion, or substitution of nucleotides, so long as the sequence provides for functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide, as operably linked components in the direction of transcription, at least regulatory elements including a transcription initiation region, a DNA of interest (e.g., a polynucleotide encoding an inhibitory RNA agent of the present disclosure), and a transcription termination region.

[0101] The terms "adeno-associated virus inverted terminal repeat" and "AAV ITR" refer to art-recognized regions flanking each end of the AAV genome that function together in cis as an origin of DNA replication and as a viral packaging signal. The AAV ITRs, together with the AAV rep coding region, provide for efficient excision and integration of the polynucleotide sequence intervening between the two adjacent ITRs into the mammalian genome. The polynucleotide sequences of the AAV ITR regions are known. As used herein, "AAV ITR" does not necessarily include the wild-type polynucleotide sequence, which may be altered, for example, by the insertion, deletion, or substitution of nucleotides. Furthermore, the AAV ITR includes, among others, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh 10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV The vector may be derived from any of several AAV serotypes, including, but not limited to, 2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16. Furthermore, the 5' and 3' ITRs flanking a selected polynucleotide sequence in an AAV vector need not be identical or derived from the same AAV serotype or isolate, so long as they function as intended, e.g., allowing for excision and rescue of the sequence of interest from the host cell genome or vector, and allowing for integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell.Furthermore, AAV ITR includes, among others, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh 10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV The vector may be derived from any of several AAV serotypes, including, but not limited to, 2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16.

[0102] The terms "antisense oligonucleotide" and "ASO" refer to an inhibitory polynucleotide that can hybridize to a target mRNA molecule (e.g., Grik2 mRNA) through complementary base pairing and inhibit its expression by destabilizing and degrading the mRNA or inhibiting translation.

[0103] The term "cDNA" refers to a nucleic acid sequence that is the DNA equivalent of an mRNA sequence (i.e., with uridines substituted for thymidines). Generally, the terms cDNA and mRNA can be used interchangeably with respect to a particular gene (e.g., the Grik2 gene), as one of skill in the art will understand that a cDNA sequence is the same as an mRNA sequence, except that uridines are read as thymidines. Furthermore, when reference is made to DNA sequences encoding the antisense constructs disclosed herein or the RNA transcripts encoded thereby, the terms "DNA" and "RNA" can be used interchangeably to refer to the antisense sequence, unless otherwise indicated by context. Furthermore, certain DNA sequences disclosed herein (e.g., those encoding Grik2 antisense sequences) may include RNA nucleotides, in which case the sequence may be referred to as a "DNA sequence" or an "RNA sequence" in its entirety.

[0104] The term "coding sequence" corresponds to a nucleic acid sequence of an mRNA molecule that encodes a protein or a portion thereof. Relatedly, a "non-coding sequence" corresponds to a nucleic acid sequence of an mRNA molecule that does not encode a protein or a portion thereof. Non-limiting examples of non-coding sequences include 5' and 3' untranslated regions (UTRs), introns, polyA tails, promoters, enhancers, terminators, and other cis-regulatory sequences.

[0105] The term "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of a polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with a polynucleotide comprising the second nucleotide sequence under specified conditions. Such conditions can be, for example, stringent conditions, which can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, can be applied. Methods for determining the most appropriate set of conditions for testing the complementarity of two sequences, depending on the end use of the hybridized nucleotide or nucleoside, are well known in the art.

[0106] As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from unnatural and alternative nucleotides, so long as the above requirements for hybridization are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing. Complementary sequences between polynucleotides and target sequences described herein include base pairing between a polynucleotide comprising a first nucleotide sequence and a polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to one another. When a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be perfectly complementary, or they can form one or more, but generally no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatched base pairs upon hybridization to a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate use, e.g., binding to and inhibiting expression of mRNA, such as Grik2 mRNA. For example, a polynucleotide is complementary to at least a portion of an mRNA of interest if the sequence is substantially complementary to an uninterrupted portion of the mRNA of interest.

[0107] The term "region of complementarity" refers to a region of an inhibitory polynucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., target sequence), or processed mRNA so as to prevent expression of an endogenous gene (e.g., Grik2). Where the region of complementarity is not perfectly complementary to the target sequence, mismatches can be in internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus of the inhibitory polynucleotide.

[0108] The terms "conservative amino acid substitution," "conservative substitution," and "conservative variation" refer to the substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized for each of the 20 naturally occurring amino acids in Table 1 below. [Table 1]

[0109] From this table, it can be seen that conservative amino acid families include: (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is one that substitutes one amino acid for a member of the same amino acid family (e.g., substitution of Ser for Thr or Lys for Arg).

[0110] The phrase "contacting a cell with an inhibitory polynucleotide," such as an inhibitory polynucleotide disclosed herein, includes contacting a cell by any possible means. Contacting a cell with an inhibitory polynucleotide includes contacting a cell with the inhibitory polynucleotide in vitro or contacting a cell with the inhibitory polynucleotide in vivo. Contacting a cell with an inhibitory polynucleotide can also mean contacting a cell with a nucleic acid vector encoding the inhibitory polynucleotide or a pharmaceutical composition containing the same. Contacting can be direct or indirect. Thus, for example, the inhibitory polynucleotide can be physically contacted with the cell by the individual performing the method, or the inhibitory polynucleotide agent can be placed in conditions that allow or cause it to subsequently contact the cell. Contacting a cell in vitro can be performed, for example, by incubating the cell with the inhibitory polynucleotide. Contacting cells in vivo can be accomplished, for example, by injecting an inhibitory polynucleotide into or near the tissue in which the cells are located, or by injecting an inhibitory polynucleotide agent into another area, such as the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the contacted cells are located. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can be contacted with an inhibitory polynucleotide in vitro and then transplanted into a subject.

[0111] Contacting a cell with an inhibitory polynucleotide includes "introducing" or "delivering the inhibitory polynucleotide to a cell" by facilitating or achieving uptake or absorption into the cell. Absorption or uptake of the inhibitory polynucleotide or the nucleic acid vector encoding it can occur by unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introducing the inhibitory polynucleotide into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the inhibitory polynucleotide can be injected at a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. In another example, the inhibitory polynucleotide can be introduced into a cell by transduction, such as with a viral vector encoding the inhibitory polynucleotide. The viral vector can undergo cellular processing (e.g., cellular internalization, encapsidation, transcription of the inhibitory polynucleotide, and processing by Drosha and Dicer) to express the encoded inhibitory polynucleotide. Additional approaches are described herein below and / or known in the art.

[0112] The terms "disrupt expression," "inhibit expression," or "reduce expression" with respect to a gene (e.g., Grik2) refer to preventing or reducing the formation of a functional gene product (e.g., GluK2 protein). A gene product is functional if it performs its normal (wild-type) function. Disruption of a gene prevents or reduces the expression of the functional protein encoded by the gene. The disrupted gene can be disrupted, for example, by an interfering RNA molecule (e.g., ASO), such as those described herein.

[0113] The terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to effect beneficial or desired results, including clinical results, when administered to a subject, including a mammal, e.g., a human. Thus, an "effective amount" or its equivalents will depend on the context in which it is being applied. For example, in the context of treating temporal lobe epilepsy (TLE), an "effective amount" or equivalent will be the amount of a composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as the given drug, pharmaceutical formulation, route of administration, the type and severity of the disease or disorder, the identity of the subject (e.g., age, sex, weight), the host being treated, and / or, in the case of epilepsy, the size of the epileptic focus (e.g., brain volume), but can nevertheless be determined according to methods well known in the art. Also, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition, vector construct, viral vector, or cell of the present disclosure can be readily determined by methods known in the art. Dosage regimens can be adjusted to provide the optimal therapeutic response.

[0114] The term "epilepsy" refers to one or more neurological disorders that clinically manifest as recurrent epileptic seizures. Epilepsy can be classified according to electroclinical syndromes according to the International League Against Epilepsy (ILAE) classification and terminology (Berg et al., 2010). These syndromes are classified by age at onset, specific constellations (surgical syndromes), and structural-metabolic causes, such as: (A) Age of onset: (i) In the neonatal period, these include benign familial neonatal epilepsy (BFNE), early myoclonic encephalopathy (EME), and Ohtahara syndrome; (ii) In infancy, these include infantile epilepsy with migratory focal seizures, West syndrome, myoclonic epilepsy in infancy (MEI), and benign infantile epilepsy. , benign familial infantile epilepsy, Dravet syndrome, myoclonic encephalopathy in non-progressive disorders; (iii) in childhood, febrile seizures plus (FS+), Panayiotopoulos syndrome, epilepsy with myoclonic atonic seizures (previously static), benign epilepsy with centrotemporal spikes (BECTS), autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), late-onset childhood occipital epilepsy (Gastaut type), epilepsy with myoclonic absence seizures, Lennox-Gastaut syndrome (iv) in adolescence and adulthood, these include juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), epilepsy with generalized tonic-clonic seizures only, progressive myoclonic epilepsy (PME), autosomal dominant epilepsy with auditory features (ADEAF), and other familial temporal lobe epilepsies. (v) Variable age onset includes familial focal epilepsy with various foci (from childhood to adulthood), reflex epilepsy; (B) Specific constellations (surgical syndromes) include mesial temporal lobe epilepsy (MTLE), Rasmussen syndrome, elastic seizures with hypothalamic hamartoma, hemiplegia-hemiplegia-epilepsy; (C) Epilepsy organized due to structural metabolic causes includes malformations of cortical development (hemimegalencephaly, heterotopia, etc.).), neurocutaneous syndromes (tuberous sclerosis complex and Sturge-Weber), tumors, infections, trauma, hemangiomas, perinatal injuries, and stroke. The term "refractory epilepsy" refers to epilepsy that is resistant to pharmaceutical treatment, i.e., current pharmaceutical treatments do not allow for effective treatment of the patient's disease (see, e.g., Dario J. Englot et al., 2013).

[0115] The term "exon" refers to a region within the coding region of a gene (e.g., the Grik2 gene), the nucleotide sequence of which determines the amino acid sequence of the corresponding protein. The term "exon" also refers to the corresponding region of RNA transcribed from a gene. Exons are transcribed into pre-mRNA and may be included in mature mRNA depending on alternative splicing of the gene. After processing, exons included in mature mRNA are translated into protein. The sequence of the exon determines the amino acid composition of the protein. Alternatively, exons included in mature mRNA may be non-coding (e.g., exons that are not translated into protein).

[0116] The term "expression," when used in reference to gene or nucleic acid expression, refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include mRNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins (e.g., GluK2) that are modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, myristoylation, and glycosylation.

[0117] The term "expression" refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of the RNA transcript (e.g., by splicing, editing, 5'-capping, and / or 3'-end processing); (3) the translation of the RNA into a polypeptide or protein; and (4) the post-translational modification of the polypeptide or protein. Expression of a gene of interest in a subject can be determined, for example, by detecting a decrease or increase in the amount or concentration of mRNA encoding the corresponding protein in a sample obtained from the subject (e.g., as assessed using RNA detection procedures described herein or known in the art, e.g., quantitative polymerase chain reaction (qPCR) and RNA-seq techniques), a decrease or increase in the amount or concentration of the corresponding protein (e.g., as assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or a decrease or increase in the activity of the corresponding protein (e.g., in the case of an ion channel, as assessed using electrophysiological methods described herein or known in the art).

[0118] The term "GluK2," also known as "GluR6," "GRIK2," "MRT6," "EAA4," or "GluK6," refers to the glutamate ionotropic receptor kainate subunit 2 protein as designated in the currently used IUPHAR nomenclature (Collingridge, G.L., Olsen, R.W., Peters, J., Spedding, M., 2009. A nomenclature for ligand-gated ion channels. Neuropharmacology 56, 2-5). The terms "GluK2-containing KAR," "GluK2 receptor," "GluK2 protein," and "GluK2 subunit" may be used interchangeably throughout and generally refer to the protein encoded by or expressed by the Grik2 gene.

[0119] The terms "guide strand" and "guide sequence" refer to a component of a stem-loop RNA structure (e.g., shRNA or microRNA) located in either the 5' or 3' stem-loop arm of the stem-loop structure, wherein the guide strand / sequence comprises a Grik2 mRNA antisense sequence (e.g., any one of SEQ ID NOs: 16-30, 63-79, 109-120, 139-144, 230-233, and 242-245, or a variant thereof having at least 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 16-30, 63-79, 109-120, 139-144, 230-233, and 242-245) that can bind to Grik2 mRNA and inhibit its expression. The guide strand / sequence may also include additional sequences, such as spacer or linker sequences. The guide sequence may be complementary or substantially complementary (e.g., with no more than 7, 6, 5, 4, 3, 2, or 1 mismatch) to the passenger strand / sequence of the stem-loop RNA structure.

[0120] The term "ionotropic glutamate receptor" includes members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and kainate receptor (KAR) classes. Functional KARs can be assembled into tetrameric assemblies from homomeric or heteromeric combinations of five subunits, designated GluK1, GluK2, GluK3, GluK4, and GluK5 (Reiner et al., 2012). The target of the present disclosure, in some instances, is the KAR complex composed of GluK2 and GluK5. Given the observation that the GluK5 subunit alone does not form functional homomeric channels, inhibiting expression of the Grik2 gene is sufficient to abolish GluK2 / GluK5 kainate receptor function.

[0121] "Expression inhibitor" refers to an agent (e.g., an inhibitory RNA agent of the present disclosure) that has the biological effect of inhibiting or reducing the expression of a gene, e.g., the Grik2 gene. Inhibiting the expression of a gene, e.g., the Grik2 gene, typically results in a reduction or even elimination of the gene product (protein, e.g., GluK2 protein) in the target cell or tissue, although various levels of inhibition can be achieved. Inhibition or reduction of expression is typically referred to as knockdown.

[0122] The term "isolated polynucleotide" refers to an isolated molecule comprising two or more covalently linked nucleotides. Such covalently linked nucleotides may also be referred to as nucleic acid molecules. Generally, an "isolated" polynucleotide refers to a polynucleotide that is artificial, chemically synthesized, purified, and / or heterologous with respect to the nucleic acid sequence from which it is derived.

[0123] The term "microRNA" refers to short (e.g., typically approximately 22 nucleotides) sequences of non-coding RNA that regulate mRNA translation and thus influence the abundance of target proteins. While some microRNAs are transcribed from a single monocistronic gene, others are transcribed as part of a polycistronic gene cluster. The structure of a microRNA may include a hairpin sequence that includes 5' and 3' flanking sequences, a stem sequence, and a loop sequence. During intracellular processing, the immature microRNA is cleaved by Drosha, which cleaves the 5' and 3' flanking sequences. The microRNA molecule then translocates from the nucleus to the cytoplasm, where it undergoes cleavage of the loop region by Dicer. The biological effects of microRNAs are exerted at the level of translational regulation through binding to a region of the mRNA molecule, typically the 3' untranslated region, resulting in mRNA cleavage, degradation, destabilization, and / or less efficient translation. Binding of a microRNA to its target is generally mediated by a short (e.g., 6-8 nucleotide) "seed region / sequence" within the hairpin sequence of the microRNA. Throughout this disclosure, the term siRNA can include its equivalent miRNA, such that the miRNA contains the same bases that share homology with the target (e.g., within the seed region) as the equivalent siRNA. As described herein, a microRNA can be a non-naturally occurring microRNA, such as a microRNA having one or more heterologous nucleic acid sequences.

[0124] The term "nucleotide" is defined as a modified or naturally occurring deoxyribonucleotide or ribonucleotide. Nucleotides typically include purines and pyrimidines, including thymidine, cytidine, guanosine, adenosine, and uridine. As used herein, the term "inhibitory polynucleotide" is defined as an oligomer of the nucleotides defined above or modified nucleotides disclosed herein. The term "inhibitory polynucleotide" refers to a 3'-5' or 5'-3' oriented nucleic acid sequence that may be single-stranded or double-stranded. Inhibitory polynucleotides used in the context of this disclosure may be, inter alia, DNA or RNA. This term may also include "inhibitory polynucleotide analogs," which refer to inhibitory polynucleotides having, for example, (i) modified backbone structures, e.g., backbones other than the standard phosphodiester linkages found in naturally occurring oligo- and polynucleotides, and (ii) optionally, modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties. Inhibitory polynucleotide analogs support bases capable of hydrogen bonding to standard polynucleotide bases through Watson-Crick base pairing, and the analog backbone presents the bases in a manner that allows for such hydrogen bonding in a sequence-specific manner between the inhibitory polynucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In particular, analogs have a substantially uncharged phosphorus-containing backbone. A substantially uncharged phosphorus-containing backbone in an inhibitory polynucleotide analog is a backbone in which the majority of the subunit linkages, e.g., 50-100%, typically at least 60-100%, 75%, or 80% of its linkages, are uncharged and contains a single phosphorus atom. Furthermore, the term "inhibitory polynucleotide" can include inhibitory polynucleotide sequences that are inverted relative to their normal orientation for transcription and thus correspond to an RNA or DNA sequence complementary to a target gene mRNA molecule expressed in a host cell. Antisense guide strands can be constructed in several different ways, as long as they are capable of disrupting expression of the target gene.For example, an antisense guide strand can be constructed by reverse-complementing the coding region (or a portion thereof) of a target gene to its normal orientation for transcription, allowing transcription of its complement (e.g., RNAs encoded by an antisense gene and a sense gene can be complementary). The inhibitory polynucleotide need not have the same intron or exon pattern as the target gene; non-coding segments of the target gene can be equally effective in achieving antisense inhibition of target gene expression, as can coding segments such as ASOs. In some cases, the inhibitory RNA has the same exon pattern as the target gene.

[0125] The inhibitory polynucleotide may be a polynucleotide that encodes Grik2 mRNA (e.g., The nucleic acid sequence may be of any length that allows targeting and hybridization to a target gene (which may be fully or substantially complementary to at least one region of an mRNA), and may range from about 10 to 50 base pairs in length, e.g., about 15 to 50 base pairs in length or about 18 to 50 base pairs in length, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 26, 15 to 28, 15 to 29, 15 to 30, 15 to 31, 15 to 32, 15 to 33, 15 to 34, 15 to 35, 15 to 36, 15 to 37, 15 to 38, 15 to 39, 26 to 40, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19 The length may be from 1 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

[0126] The terms "passenger strand" and "passenger sequence" refer to Grik2 This refers to a component of a stem-loop RNA structure (e.g., shRNA or microRNA) located in either the 5' or 3' stem-loop arm of the stem-loop structure, which comprises a sequence complementary or substantially complementary (e.g., having no more than 7, 6, 5, 4, 3, 2, or 1 mismatch) to an mRNA antisense sequence (e.g., any one of SEQ ID NOs: 16 to 30, 63 to 79, 109 to 120, 139 to 144, 230 to 233, and 242 to 245, or a variant thereof having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 16 to 30, 63 to 79, 109 to 120, 139 to 144, 230 to 233, and 242 to 245). The passenger strand / sequence may also comprise additional sequences, such as spacer or linker sequences, etc. The passenger sequence may be complementary or substantially complementary to the guide strand / sequence of the stem-loop RNA structure.

[0127] The term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Certain plasmids can replicate autonomously in a host cell into which they are introduced (e.g., bacterial plasmids with a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Certain plasmids can direct the expression of genes to which they are operably linked. As used herein, "gene" refers to a polynucleotide that encodes a protein, microRNA, siRNA, shRNA, or shmiRNA and further comprises one or more regulatory sequences (e.g., a promoter, enhancer, intron, or termination sequence, among others).

[0128] The term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of a polynucleotide. Exemplary promoters suitable for use in the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which is incorporated herein by reference as it relates to nucleic acid regulatory elements. Additionally, the term "promoter" can refer to synthetic promoters, which are regulatory DNA sequences that do not naturally occur in biological systems. Synthetic promoters contain portions of naturally occurring promoters combined with non-naturally occurring polynucleotide sequences and can be optimized to express recombinant DNA using a variety of polynucleotides, vectors, and target cell types.

[0129] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Well-recognized conventional methods can be used to determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to a given nucleic acid or amino acid sequence B (or, alternatively, a given nucleic acid or amino acid sequence, A, can be said to have a certain percent sequence identity to a given nucleic acid or amino acid sequence B, to a given nucleic acid or amino acid sequence B, or to a given nucleic acid or amino acid sequence B) is calculated as follows: Multiply 100 by (fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A. Regardless of the percent sequence identity between a candidate sequence and a reference polynucleotide or polypeptide sequence, the candidate sequence retains at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% of the function of the reference polynucleotide or polypeptide sequence (e.g., the ability to reduce levels of Grik2 mRNA as defined herein or expression levels of GluK2 protein as defined herein).

[0130] The term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response and other problematic complications commensurate with a reasonable benefit / risk ratio.

[0131] As used herein, the term "pharmaceutical composition" refers to a composition containing a compound described herein (e.g., an inhibitory nucleic acid molecule (e.g., RNA) or a vector comprising same) formulated with a pharmaceutically acceptable excipient, and may, in some cases, be manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for treating a mammalian disease. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., tablet, capsule, caplet, gelcap, or syrup), topical administration (e.g., as a cream, gel, lotion, or ointment), intravenous administration (e.g., as a sterile solution in a solvent system that is free of particulate emboli and suitable for intravenous use), intrathecal injection, intraventricular injection, intraparenchymal injection, or any other pharmaceutically acceptable formulation.

[0132] A "pharmaceutically acceptable excipient" refers to any ingredient other than a compound described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has the properties of being substantially non-toxic and non-inflammatory in a patient. Excipients can include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-formers or coatings, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0133] The compounds described herein (e.g., inhibitory nucleic acid molecules (e.g., RNA) and vectors containing same) may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or, in the case of the compounds described herein in their acidic form, salts may be prepared from inorganic or organic bases. In many cases, compounds are prepared or used as pharmaceutically acceptable salts, prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, as well as methods for preparing suitable salts, are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, Examples of suitable salts include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0134] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signal sequences) that control the transcription or translation of a gene. Such regulatory sequences are described, for example, in Perdew et al., Regulation of Gene Expression (Humana Press, New York, NY, (2014)), which is incorporated herein by reference.

[0135] The term "target" or "targeting" refers to the ability of an inhibitory nucleic acid molecule (e.g., RNA), such as an inhibitory RNA agent described herein, to specifically bind to the Grik2 gene or mRNA encoding the GluK2 protein through complementary base pairing.

[0136] The terms "small interfering RNA" and "siRNA" refer to inhibitory polynucleotides comprising double-stranded nucleic acids, each strand of which comprises RNA, RNA analog(s), or RNA and DNA. An siRNA molecule can contain 19-23 nucleotides (e.g., 21 nucleotides). An siRNA typically has a 2-bp overhang at the 3' end of each strand such that the duplex region in the siRNA contains 17-21 nucleotides (e.g., 19 nucleotides). Typically, the antisense strand of an siRNA is sufficiently complementary to the target sequence of a target gene / RNA. An siRNA molecule acts within the RNA interference pathway, binding to a target mRNA (e.g., Grik2 mRNA) and degrading the mRNA through Dicer-mediated mRNA cleavage, thereby inhibiting mRNA expression. Throughout this disclosure, the term siRNA is meant to include its equivalent miRNA, as an miRNA contains the same bases that share homology with the target as its equivalent siRNA.

[0137] The terms "short hairpin RNA" and "shRNA" refer to inhibitory polynucleotides comprising a 50-100 nucleotide single-stranded RNA that forms a stem-loop structure in cells, including a 5-30 nucleotide loop region, and a 15-50 nucleotide longer complementary RNA on either side of the loop region, forming a double-stranded stem through base pairing between the complementary RNA sequences. In some cases, an additional 1-500 nucleotides are included before and after each complementary strand that forms the stem. For example, shRNAs generally require a specific sequence 3' of the hairpin to terminate transcription by RNA polymerase. Such shRNAs generally bypass Drosha processing due to the short 5' and 3' flanking sequences. Other shRNAs, such as "shRNA-like microRNAs" transcribed by RNA polymerase II, contain longer 5' and 3' flanking sequences and require Drosha processing in the nucleus. The cleaved shRNA is then transported from the nucleus to the cytosol, where it is further cleaved by Dicer. Like siRNA, shRNA binds to target mRNA in a sequence-specific manner, cleaves and destroys the target mRNA, and suppresses the expression of the target mRNA.

[0138] As used herein, the terms "specifically hybridize" and "specifically bind" refer to a polynucleotide that has a sufficient degree of complementarity between the polynucleotide and a target nucleic acid (e.g., Grik2 mRNA) to induce a desired effect (e.g., reduction or inhibition of GluK2 expression from Grik2 mRNA), but exhibits minimal or no effect on non-target nucleic acids. Specific hybridization or binding can occur under physiological conditions. For example, specific hybridization or binding occurs when the number of nucleobases in a polynucleotide (e.g., an antisense polynucleotide) that are complementary to the nucleobases of a corresponding target nucleic acid (e.g., an mRNA sequence) promotes annealing of the polynucleotide to the target nucleic acid but not to a non-target nucleic acid (e.g., complementarity corresponding to, for example, a percent sequence identity of 80% or more (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) of the binding portion of the polynucleotide to the target nucleic acid). Those skilled in the art will understand that in such situations, the nucleic acid sequence in the polynucleotide (e.g., antisense oligomer) and the nucleic acid sequence in the target nucleic acid will have a high degree of complementarity (e.g., at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementarity to a defined number of polynucleotides (e.g., about 7-22 nucleic acid bases), etc.).

[0139] The terms "subject" and "patient" refer to an animal (e.g., a mammal such as a human). A subject treated according to the methods described herein may be a subject diagnosed with epilepsy (e.g., TLE) or at risk for developing the condition. Diagnosis can be made by any method or technique known in the art. A subject treated according to the present disclosure may have been subjected to standard testing, or may be identified as at risk without testing due to the presence of one or more risk factors associated with the disease or condition.

[0140] The term "temporal lobe epilepsy" or "TLE" refers to a chronic neurological condition characterized by chronic and recurrent seizures (epilepsy) originating in the temporal lobe of the brain. This disease differs from acute seizures in naive brain tissue because TLE is characterized by morphofunctional reorganization of neuronal networks and recurrent mossy fiber sprouting from granule cells in the dentate gyrus of the hippocampus, whereas acute seizures in naive tissue do not precipitate such circuit-specific reorganization. TLE can result from the emergence of epileptogenic foci in one or both hemispheres of the brain.

[0141] The terms "transduction" and "transduction" refer to a method of introducing nucleic acid material (e.g., a vector, such as a viral vector construct, or a portion thereof) into a cell, followed by expression in the cell of a polynucleotide encoded by the nucleic acid material (e.g., a vector construct or a portion thereof).

[0142] The term "therapy / treatment" or "treating" refers to both preventative and prophylactic treatment, as well as curative or disease-modifying treatment, and includes treatment of patients at risk of or suspected of having a disease, as well as patients diagnosed with a disease or condition. Treatment also includes the suppression of clinical recurrence. Treatment may be administered to a subject with a medical disorder or who may ultimately acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disease or recurrent disease, or to extend the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment for a disease, e.g., an administration pattern used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the therapeutic regimen. The induction regimen can use (partially or entirely) a "loading regimen," which can include administering a higher dose of drug than the physician uses during the maintenance regimen, administering the drug more frequently than the physician uses during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep a patient in remission over an extended period of time (months or years). A maintenance regimen can use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., disease manifestation)).

[0143] The term "vector" includes nucleic acid vectors, e.g., DNA vectors, e.g., plasmids, RNA vectors, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering exogenous polynucleotides or polynucleotides encoding proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 011026, which is incorporated herein by reference as it relates to vectors suitable for expressing nucleic acid material of interest. Expression vectors suitable for use in the compositions and methods described herein contain polynucleotide sequences used for expression of heterologous nucleic acid material (e.g., ASO) in mammalian cells as well as, for example, additional sequence elements. Particular vectors that can be used for expression of the inhibitory nucleic acid (e.g., RNA) agents described herein include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for expression of the inhibitory nucleic acid (e.g., RNA) agents disclosed herein contain polynucleotide sequences that enhance the translation rate of these polynucleotides or improve the stability or nuclear export of nucleic acids (e.g., RNA) resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRES, and polyadenylation signal sequence sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0144] As used herein, the term "variant" refers to a polynucleotide, such as an inhibitory polynucleotide sequence of the present disclosure or its complement (e.g., a substantial or perfect complement thereof), resulting from the rational inclusion of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide modifications (substitutions, insertions, deletions, or mismatches) relative to a starting sequence (e.g., a reference sequence). Such modifications may improve at least one characteristic (e.g., biological function) of the polynucleotide (e.g., improved RISC loading or retention of the guide strand, decreased RISC loading or retention of the passenger strand, or an increased ratio of guide-to-strand production, and improved inhibition of the target nucleic acid sequence). [Brief explanation of the drawings]

[0145] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0146] [Figure 1A]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1B]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1C]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1D]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1E]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1F]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1G]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1H]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1I]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1J]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1K]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1L]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1M]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1N]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1O]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1P]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1Q]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1R]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1S]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1T]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1U]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1V]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 1W]Figures 1A-1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) or its variants incorporated into an endogenous (E)-miR-30 microRNA scaffold. The stem-loop structures contain, from 5' to 3', a guide strand containing the GI antisense sequence or its rationally designed variants (SEQ ID NOs: 17-30, 230-233, and 242-245), an E-miR-30 loop sequence, and a passenger sequence (SEQ ID NO: 31) or its rationally designed variants (SEQ ID NOs: 32-45, 234-237, and 246-249). The starting construct (Construct A) is shown in Figure 1A. Changes to the starting construct are shown in Figures 1B-1W, respectively. Small black dots correspond to the UG wobble pair. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A delivered into induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (GlutaNeurons). [Figure 2A]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2B]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2C]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2D]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2E]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2F]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2G]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2H]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2I]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2J]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2K]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2L]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2M]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2N]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2O]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2P]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 2Q]Figures 2A-2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures include the G9 antisense sequence or its rationally designed variants (SEQ ID NOs: 64-79), the E-miR-124-3 loop sequence, and a guide strand containing a passenger sequence (SEQ ID NO: 80) or its rationally designed variants (SEQ ID NOs: 81-96). The starting construct (Construct B) is shown in Figure 2A. Changes to the starting construct are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A-2I feature stem-loop structures containing, from 5' to 3', the passenger strand, the loop sequence, and the guide strand, while Figures 2J-2Q feature stem-loop structures containing, from 5' to 3', the guide strand, the loop sequence, and the passenger strand. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from starting construct B delivered to GlutaNeurons. [Figure 3A]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3B] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3C]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3D] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3E]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3F] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3G]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3H] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3I]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3J] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3K]Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 3L] Figures 3A-3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) or its variants incorporated into the endogenous E-miR-124-3 microRNA scaffold. The stem-loop structures comprise, from 5' to 3', a passenger sequence (SEQ ID NO: 121) or its rationally designed variants (SEQ ID NOs: 122-132), an E-miR-124-3 loop sequence, and a guide strand containing the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 110-120). The starting construct (Construct C) is shown in Figure 3A. Changes to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct C delivered to GlutaNeurons. [Figure 4A]Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 4B] Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 4C]Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 4D] Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 4E]Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 4F] Figures 4A-4F are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or its variants incorporated into the endogenous E-miR-218-1 microRNA scaffold. The stem-loop structures contain, from 5' to 3', the MW antisense sequence or its rationally designed variants (SEQ ID NOs: 140-144), the E-miR-218-1 loop sequence, and a guide strand containing the passenger sequence (SEQ ID NO: 145) or its rationally designed variants (SEQ ID NOs: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes to the starting construct are shown in Figures 4B-4F, respectively. Small black dots correspond to UG wobble pairs. Large black dots with numbers correspond to the design criteria described in Example 1. *Drosha and Dicer cleavage sites are based on the most abundant species observed in small RNA sequencing data obtained from Starting Construct D delivered to GlutaNeurons. [Figure 5A]Figures 5A-5E are images of AAV expression constructs containing a single microRNA construct of the present disclosure. The general construct structure, from 5' to 3', features: AAV 5' ITR, hSyn1 promoter sequence, stem-loop sequence contained 5' to 3': 5' microRNA flanking sequence, 5' stem-loop arm containing either guide strand or passenger strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing either passenger strand or guide strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA) and AAV 3' ITR (Figure 5A). Figure 5B shows an AAV vector, Construct No. 102, containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, Construct No. 103, containing the stem-loop sequence of Construct No. 51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing the stem-loop sequence of construct number 39 (SEQ ID NO: 98), and Figure 5E shows an AAV vector containing the stem-loop sequence of construct number 40 (SEQ ID NO: 99). [Figure 5B]Figures 5A-5E are images of AAV expression constructs containing a single microRNA construct of the present disclosure. The general construct structure, from 5' to 3', features: AAV 5' ITR, hSyn1 promoter sequence, stem-loop sequence contained 5' to 3': 5' microRNA flanking sequence, 5' stem-loop arm containing either guide strand or passenger strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing either passenger strand or guide strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA) and AAV 3' ITR (Figure 5A). Figure 5B shows an AAV vector, Construct No. 102, containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, Construct No. 103, containing the stem-loop sequence of Construct No. 51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing the stem-loop sequence of construct number 39 (SEQ ID NO: 98), and Figure 5E shows an AAV vector containing the stem-loop sequence of construct number 40 (SEQ ID NO: 99). [Figure 5C]Figures 5A-5E are images of AAV expression constructs containing a single microRNA construct of the present disclosure. The general construct structure, from 5' to 3', features: AAV 5' ITR, hSyn1 promoter sequence, stem-loop sequence contained 5' to 3': 5' microRNA flanking sequence, 5' stem-loop arm containing either guide strand or passenger strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing either passenger strand or guide strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA) and AAV 3' ITR (Figure 5A). Figure 5B shows an AAV vector, Construct No. 102, containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, Construct No. 103, containing the stem-loop sequence of Construct No. 51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing the stem-loop sequence of construct number 39 (SEQ ID NO: 98), and Figure 5E shows an AAV vector containing the stem-loop sequence of construct number 40 (SEQ ID NO: 99). [Figure 5D]Figures 5A-5E are images of AAV expression constructs containing a single microRNA construct of the present disclosure. The general construct structure, from 5' to 3', features: AAV 5' ITR, hSyn1 promoter sequence, stem-loop sequence contained 5' to 3': 5' microRNA flanking sequence, 5' stem-loop arm containing either guide strand or passenger strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing either passenger strand or guide strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA) and AAV 3' ITR (Figure 5A). Figure 5B shows an AAV vector, Construct No. 102, containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, Construct No. 103, containing the stem-loop sequence of Construct No. 51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing the stem-loop sequence of construct number 39 (SEQ ID NO: 98), and Figure 5E shows an AAV vector containing the stem-loop sequence of construct number 40 (SEQ ID NO: 99). [Figure 5E]Figures 5A-5E are images of AAV expression constructs containing a single microRNA construct of the present disclosure. The general construct structure, from 5' to 3', features: AAV 5' ITR, hSyn1 promoter sequence, stem-loop sequence contained 5' to 3': 5' microRNA flanking sequence, 5' stem-loop arm containing either guide strand or passenger strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing either passenger strand or guide strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA) and AAV 3' ITR (Figure 5A). Figure 5B shows an AAV vector, Construct No. 102, containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, Construct No. 103, containing the stem-loop sequence of Construct No. 51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing the stem-loop sequence of construct number 39 (SEQ ID NO: 98), and Figure 5E shows an AAV vector containing the stem-loop sequence of construct number 40 (SEQ ID NO: 99). [Figure 6A] Figures 6A and 6B are images of AAV expression constructs containing concatemeric constructs of the present disclosure. Figure 6A shows a dual microRNA AAV vector, Construct No. 100, containing the stem-loop sequences of Construct No. 3 (SEQ ID NO: 4) and Construct No. 51 (SEQ ID NO: 135), with Construct No. 3 located 5' to Construct No. 51. Figure 6B shows a concatemeric AAV vector containing the stem-loop sequences of Construct No. 3 (SEQ ID NO: 4) and Construct No. 51 (SEQ ID NO: 135), with Construct No. 3 located 3' to Construct No. 51. [Figure 6B]Figures 6A and 6B are images of AAV expression constructs containing concatemeric constructs of the present disclosure. Figure 6A shows a dual microRNA AAV vector, Construct No. 100, containing the stem-loop sequences of Construct No. 3 (SEQ ID NO: 4) and Construct No. 51 (SEQ ID NO: 135), with Construct No. 3 located 5' to Construct No. 51. Figure 6B shows a concatemeric AAV vector containing the stem-loop sequences of Construct No. 3 (SEQ ID NO: 4) and Construct No. 51 (SEQ ID NO: 135), with Construct No. 3 located 3' to Construct No. 51. [Figure 7] Figure 7 is a graph showing the relative expression levels of human Grik2 mRNA, as quantified by RT-qPCR, in SH-SY5Y cells transfected as described in Example 3. n=4 for all groups. One-way ANOVA, Dunnett's multiple comparison test (vs. siNegative). **p<0.001; error bars: standard deviation. Key: RNAiMAX = transfection reagent only; siNegative = siRNA negative control; siPositive = siRNA positive control; A, C, D = constructs A, C, and D, respectively; #1, #2, #3, #4, #39, #40, #50, and #51 = constructs #1, #2, #3, #4, #39, #40, #50, and #51, respectively. [Figure 8A]Figures 8A and 8B are graphs showing the expression of miRNAs GI and MW and GLUK2 protein levels, respectively, in mouse cortical neurons (MCNs) after transduction with AAV vectors. Figure 8A shows quantification of GI and MW by stem-loop RT-qPCR. The y-axis represents the number of GI or MW miRNA molecules per 10 pg of total RNA expressed in cells transduced with the following AAV vectors: (left to right) RNA null vector (Ctrl), double-miRNA concatemer, construct number 100 (SEQ ID NO: 256) containing the stem-loop sequence of construct number 3 (SEQ ID NO: 4) located 5' to construct number 51 (SEQ ID NO: 135), double-miRNA concatemer, construct number 101 (SEQ ID NO: 257) containing the stem-loop sequence of construct number 51 located 5' to construct number 3, a single construct containing only the GI sequence (SEQ ID NO: 252), and a single construct containing only the MW sequence (SEQ ID NO: 253). Figure 8B shows GLUK2 protein levels quantified by immunoblot. Control wells were treated with AAV9.hSyn.GFP, an RNA null control vector, or were untreated. The figure shows the fold change in GLUK2 / GLUK3 expression normalized to beta-actin versus the AAV9.hSyn.GFP control for each condition. **P<0.01. [Figure 8B]Figures 8A and 8B are graphs showing the expression of miRNAs GI and MW and GLUK2 protein levels, respectively, in mouse cortical neurons (MCNs) after transduction with AAV vectors. Figure 8A shows quantification of GI and MW by stem-loop RT-qPCR. The y-axis represents the number of GI or MW miRNA molecules per 10 pg of total RNA expressed in cells transduced with the following AAV vectors: (left to right) RNA null vector (Ctrl), double-miRNA concatemer, construct number 100 (SEQ ID NO: 256) containing the stem-loop sequence of construct number 3 (SEQ ID NO: 4) located 5' to construct number 51 (SEQ ID NO: 135), double-miRNA concatemer, construct number 101 (SEQ ID NO: 257) containing the stem-loop sequence of construct number 51 located 5' to construct number 3, a single construct containing only the GI sequence (SEQ ID NO: 252), and a single construct containing only the MW sequence (SEQ ID NO: 253). Figure 8B shows GLUK2 protein levels quantified by immunoblot. Control wells were treated with AAV9.hSyn.GFP, an RNA null control vector, or were untreated. The figure shows the fold change in GLUK2 / GLUK3 expression normalized to beta-actin versus the AAV9.hSyn.GFP control for each condition. **P<0.01. [Figure 9] 9 is a graph showing Grik2 mRNA expression quantified by RNA sequencing in iPSC-derived GlutaNeuron cells after transduction with either an RNA null vector (Ctrl) or an AAV encoding Construct No. 51 (SEQ ID NO: 135), a double-miRNA concatemer containing the stem-loop sequence of Construct No. 3 (SEQ ID NO: 4) located 5' to Construct No. 100 (SEQ ID NO: 256). TPM, transcripts per million. **FDR (P adj) < 0.01. [Figure 10A]Figures 10A and 10B are graphs displaying epileptic activity in adjacent human brain slices from two patients with temporal lobe epilepsy (TLE). Brain slices from one patient were recorded under hyperexcitable conditions, while brain slices from the other patient were recorded under physiological conditions. Figure 10A shows adjacent organotypic hippocampal slices from a TLE patient recorded in the presence of 4-AP / gabazine. The left side of the panel shows raw traces of seizure events recorded after transduction with a control vector (AAV 9.hSyn.GFP). The right side of the panel shows raw traces showing epileptiform discharges after transduction with construct #100 (AAV9.hSyn. construct #3 / construct #51; SEQ ID NO: 256). Compared to controls, construct #100 significantly suppressed spontaneous seizures from the TLE hippocampus ex vivo under hyperexcitable conditions. Figure 10B shows neuronal excitability in organotypic hippocampal slices from another TLE patient, where the slices were recorded under physiological conditions to record spontaneous seizure activity from an RNA null control and after transduction with Construct No. 100. Compared to controls, Construct No. 100 significantly suppressed spontaneous seizures from the TLE hippocampus ex vivo in physiological buffer conditions. [Figure 10B]Figures 10A and 10B are graphs displaying epileptic activity in adjacent human brain slices from two patients with temporal lobe epilepsy (TLE). Brain slices from one patient were recorded under hyperexcitable conditions, while brain slices from the other patient were recorded under physiological conditions. Figure 10A shows adjacent organotypic hippocampal slices from a TLE patient recorded in the presence of 4-AP / gabazine. The left side of the panel shows raw traces of seizure events recorded after transduction with a control vector (AAV 9.hSyn.GFP). The right side of the panel shows raw traces showing epileptiform discharges after transduction with construct #100 (AAV9.hSyn. construct #3 / construct #51; SEQ ID NO: 256). Compared to controls, construct #100 significantly suppressed spontaneous seizures from the TLE hippocampus ex vivo under hyperexcitable conditions. Figure 10B shows neuronal excitability in organotypic hippocampal slices from another TLE patient, where the slices were recorded under physiological conditions to record spontaneous seizure activity from an RNA null control and after transduction with Construct No. 100. Compared to controls, Construct No. 100 significantly suppressed spontaneous seizures from the TLE hippocampus ex vivo in physiological buffer conditions. [Figure 11A]Figures 11A-11C are graphs showing behavioral assessment of epilepsy-related phenotypes in the pilocarpine mouse model. Chronically epileptic mice were treated (n=5) with either an RNA null control vector (Ctrl) or construct #100 (SEQ ID NO: 256), construct #101 (SEQ ID NO: 257), a single construct containing only the GI sequence (SEQ ID NO: 252), or a single construct containing only the MW sequence (SEQ ID NO: 253), all administered at 1E+9 GC / brain. *p<0.05, **p<0.01, Mann-Whitney test. The concatemeric vectors, construct #100 and construct #101, were effective in ameliorating epilepsy-related phenotypes in the pilocarpine model in vivo. Figure 11A shows the total distance covered by chronically epileptic mice during a 10-minute exploration period in an open field box. Epileptic mice were hyperactive, traveling approximately twice the distance compared to non-epileptic mice. Thus, mice treated with the concatemer vector behaved more like non-epileptic mice and moved less after treatment. Figure 11B shows the average number of seizures per day for chronically epileptic mice treated with either the RNA null control, the first concatemer (Construct No. 100), or the second concatemer (Construct No. 101). Figure 11C shows behavioral scoring based on five animal behaviors (nesting, trembling, hair, handling, and spontaneous locomotion). The Y-axis represents the sum of the scores for the five behaviors. Control represents epileptic mice treated with the control vector. Mice treated with Construct No. 100 exhibited behavior similar to that of normal, non-epileptic mice. [Figure 11B]Figures 11A-11C are graphs showing behavioral assessment of epilepsy-related phenotypes in the pilocarpine mouse model. Chronically epileptic mice were treated (n=5) with either an RNA null control vector (Ctrl) or construct #100 (SEQ ID NO: 256), construct #101 (SEQ ID NO: 257), a single construct containing only the GI sequence (SEQ ID NO: 252), or a single construct containing only the MW sequence (SEQ ID NO: 253), all administered at 1E+9 GC / brain. *p<0.05, **p<0.01, Mann-Whitney test. The concatemeric vectors, construct #100 and construct #101, were effective in ameliorating epilepsy-related phenotypes in the pilocarpine model in vivo. Figure 11A shows the total distance covered by chronically epileptic mice during a 10-minute exploration period in an open field box. Epileptic mice were hyperactive, traveling approximately twice the distance compared to non-epileptic mice. Thus, mice treated with the concatemer vector behaved more like non-epileptic mice and moved less after treatment. Figure 11B shows the average number of seizures per day for chronically epileptic mice treated with either the RNA null control, the first concatemer (Construct No. 100), or the second concatemer (Construct No. 101). Figure 11C shows behavioral scoring based on five animal behaviors (nesting, trembling, hair, handling, and spontaneous locomotion). The Y-axis represents the sum of the scores for the five behaviors. Control represents epileptic mice treated with the control vector. Mice treated with Construct No. 100 exhibited behavior similar to that of normal, non-epileptic mice. [Figure 11C]Figures 11A-11C are graphs showing behavioral assessment of epilepsy-related phenotypes in the pilocarpine mouse model. Chronically epileptic mice were treated (n=5) with either an RNA null control vector (Ctrl) or construct #100 (SEQ ID NO: 256), construct #101 (SEQ ID NO: 257), a single construct containing only the GI sequence (SEQ ID NO: 252), or a single construct containing only the MW sequence (SEQ ID NO: 253), all administered at 1E+9 GC / brain. *p<0.05, **p<0.01, Mann-Whitney test. The concatemeric vectors, construct #100 and construct #101, were effective in ameliorating epilepsy-related phenotypes in the pilocarpine model in vivo. Figure 11A shows the total distance covered by chronically epileptic mice during a 10-minute exploration period in an open field box. Epileptic mice were hyperactive, traveling approximately twice the distance compared to non-epileptic mice. Thus, mice treated with the concatemer vector behaved more like non-epileptic mice and moved less after treatment. Figure 11B shows the average number of seizures per day for chronically epileptic mice treated with either the RNA null control, the first concatemer (Construct No. 100), or the second concatemer (Construct No. 101). Figure 11C shows behavioral scoring based on five animal behaviors (nesting, trembling, hair, handling, and spontaneous locomotion). The Y-axis represents the sum of the scores for the five behaviors. Control represents epileptic mice treated with the control vector. Mice treated with Construct No. 100 exhibited behavior similar to that of normal, non-epileptic mice. [Figure 12A]Figures 12A and 12B are graphs of distance traveled or seizure activity in pilocarpine mice treated with either an RNA null control vector (Ctrl) or Construct No. 100 (SEQ ID NO: 256). At the tested dose of 1E+10 GC / brain, Construct No. 100 was effective in reducing the hyperkinetic phenotype and seizure activity in the pilocarpine mouse model in vivo. Figure 12A shows the total distance covered by chronic epileptic mice during 10 minutes of exploration in an open field box. Chronic epileptic mice were treated with either a control vector or Construct No. 100 applied at 1E+10 GC / brain. ****p<0.0001, Mann-Whitney test. Figure 12B shows the mean daily number of seizures in chronic epileptic mice one month after treatment with either a control vector or Construct No. 100. **p<0.01, Mann-Whitney test. [Figure 12B] Figures 12A and 12B are graphs of distance traveled or seizure activity in pilocarpine mice treated with either an RNA null control vector (Ctrl) or Construct No. 100 (SEQ ID NO: 256). At the tested dose of 1E+10 GC / brain, Construct No. 100 was effective in reducing the hyperkinetic phenotype and seizure activity in the pilocarpine mouse model in vivo. Figure 12A shows the total distance covered by chronic epileptic mice during 10 minutes of exploration in an open field box. Chronic epileptic mice were treated with either a control vector or Construct No. 100 applied at 1E+10 GC / brain. ****p<0.0001, Mann-Whitney test. Figure 12B shows the mean daily number of seizures in chronic epileptic mice one month after treatment with either a control vector or Construct No. 100. **p<0.01, Mann-Whitney test. [Figure 13A]Figures 13A and 13B are graphs showing the dose-dependent reduction in hyperkinetic phenotype and seizures in pilocarpine mice treated with Construct #100. Figure 13A shows the total distance covered during 10 minutes of exploration in an open field box. Chronically epileptic mice were treated with either an RNA null control vector (Ctrl) or Construct #100 (1E+8 / 1E+9 / 1E+10 GC / brain). **p<0.01, Mann-Whitney test. The past locomotor activity of wild-type mice (WT) was assessed in a separate experiment and is shown here for comparison. Figure 13B shows the average number of seizures per day in chronically epileptic mice after treatment with either the control vector or Construct #100. [Figure 13B] Figures 13A and 13B are graphs showing the dose-dependent reduction in hyperkinetic phenotype and seizures in pilocarpine mice treated with Construct #100. Figure 13A shows the total distance covered during 10 minutes of exploration in an open field box. Chronically epileptic mice were treated with either an RNA null control vector (Ctrl) or Construct #100 (1E+8 / 1E+9 / 1E+10 GC / brain). **p<0.01, Mann-Whitney test. The past locomotor activity of wild-type mice (WT) was assessed in a separate experiment and is shown here for comparison. Figure 13B shows the average number of seizures per day in chronically epileptic mice after treatment with either the control vector or Construct #100. [Figure 14] Figure 14 is an image of a vector map containing the inhibitory polynucleotide sequence of Construct No. 100. Construct No. 100 contains a lac promoter sequence, an ampicillin resistance (AmpR) promoter sequence, and a kanamycin resistance (KanR) sequence, although other promoters and antibiotic resistance sequences (e.g., a chloramphenicol resistance sequence) can alternatively be included. DETAILED DESCRIPTION OF THE INVENTION

[0147] Detailed Description Described herein are compositions and methods for treating epilepsy, such as temporal lobe epilepsy (TLE; e.g., treatment-refractory TLE), in a subject (e.g., a mammalian subject, e.g., a human) using inhibitory polynucleotides (e.g., polynucleotides encoding inhibitory RNA agents) having modifications designed to affect (e.g., improve) RNA-induced silencing complex (RISC) loading, e.g., enhance antisense guide strand production and minimize passenger strand production, thereby facilitating greater knockdown of Grik2 mRNA and GluK2 expression and reducing the potential risk of off-target effects and toxicity induced by the passenger strand. For example, a therapeutically effective amount of an inhibitory RNA molecule (e.g., an antisense oligonucleotide (ASO), shRNA, siRNA, shmiRNA, or nucleic acid vector encoding the same, e.g., those described herein) targeting mRNA encoded by the glutamate ionotropic receptor kainate-type subunit 2 (Grik2) gene can be administered, e.g., according to the methods described herein, to treat epilepsy in a subject (e.g., a human) in need thereof. Also described herein are compositions containing a nucleic acid vector (e.g., a viral vector, e.g., an adeno-associated virus (AAV) vector) encoding an inhibitory RNA agent that targets Grik2 mRNA for the treatment of TLE.

[0148] Grik2 Grik2 is the gene encoding the ionotropic glutamate receptor subunit GluK2, which is activated by the endogenous agonist glutamate and can also be selectively activated by the agonist kainate. GluK2-containing kainate receptors (KARs), like other ionotropic glutamate receptors, exhibit rapid ligand gating by glutamate, acting by opening a cation channel pore permeable to sodium and potassium. KAR complexes can be assembled from several subunits as heteromeric or homomeric assemblies of KAR subunits. Such receptors are characterized by an extracellular N-terminus and a large peptide loop that together form the ligand-binding domain and intracellular C-terminus. The ionotropic glutamate receptor complex itself acts as a ligand-gated ion channel, mediating the passage of charged ions across neuronal membranes upon binding of glutamate. Generally, KARs are multimeric assemblies of GluK1, 2, and / or 3 (previously designated GluR5, GluR6, and GluR7, respectively), GluK4 (KA1), and GluK5 (KA2) subunits (Collingridge, Neuropharmacology. 2009 Jan;56(1):2-5). The various combinations of subunits involved in KAR complexes are often determined by RNA splicing and / or RNA editing (e.g., adenosine to inosine conversion by adenosine deaminase) of the mRNA encoding specific KAR subunits. Furthermore, such RNA modifications can affect receptor properties, such as altering the calcium permeability of the channel. Increased activity of kainate receptors is known to be epileptogenic. GluK2-containing KARs are suitable targets for modulating ionotropic glutamate receptor activity and subsequently ameliorating symptoms associated with epileptogenesis (Peret et al., 2014).

[0149] Temporal lobe epilepsy Epileptogenesis is a process that can potentially lead to the establishment of epilepsy and involves the cellular, molecular, and morphological changes that result in pathological neural network reorganization. TLE is characterized by two major types based on the anatomical origin of the epileptogenic focus. TLE originating in the medial temporal lobe (e.g., the hippocampus, parahippocampal gyrus, subiculum, and amygdala, among others) is called medial TLE (mTLE), while TLE originating in the lateral temporal lobe (e.g., the temporal neocortex) is called lateral TLE (lTLE). Additional features characteristic of TLE may include neuronal cell death in the CA1, CA3, dentate gyrus, and dentate gyrus (DG) regions of the hippocampus, reversal of GABA reversal potentials, dispersion of granule cells (GCs) in the DG, and recurrent GC-mossy fiber sprouting, which leads to the formation of pathophysiological recurrent excitatory synapses onto dentate GCs (rMF-DGC synapses).

[0150] Various causative factors have been attributed to the pathogenesis of TLE, including mesial temporal lobe sclerosis, traumatic brain injury, brain infections (e.g., encephalitis and meningitis), hypoxic brain injury, stroke, brain tumors, genetic syndromes, and febrile seizures. Because CNS plasticity depends on both developmental status and brain region-specific susceptibility, not all subjects with brain injury develop epilepsy. The hippocampus, including the DG, has been identified as a brain region particularly susceptible to TLE-causing damage and, in some cases, is associated with treatment-resistant (i.e., intractable) epilepsy (Jarero-Basulto, JJ, et al. Pharmaceuticals, 2018, 11, 17; doi:10.3390 / ph11010017). Amplification of excitatory glutamatergic signaling can promote spontaneous seizures (Kuruba, et al. Epilepsy Behav. 2009, 14(Suppl. 1), 65-73).

[0151] Without wishing to be bound by theory, abnormal rMF-DGC synapses operating through ectopic GluK2-containing KARs (Epsztein et al., 2005; Artinian et al., 2011, 2015) may play an important role in chronic seizures in TLE (Peret et al., 2014). For example, interictal spikes and seizure events (i.e., electrophysiological signatures of epileptiform brain activity) were reduced in transgenic mice lacking the GluK2 receptor subunit or in the presence of pharmacological agents that inhibit GluK2 / GluK5 receptors (Peret et al., 2014; Crepel and Mulle, 2015). While knockdown or silencing of GluK2 in transgenic animal models designed to test these theories is feasible, designing inhibitors that are selective for the GluK2 subunit and safe for use in humans is challenging. GluK subunits are structurally conserved, and their DNA coding sequences share significant homology. The complex gene expression patterns in the brain for homomeric and heteromeric ionotropic and metabotropic glutamate receptors further complicate any treatment strategy. The methods and compositions disclosed herein are suitable for treating TLE (e.g., mTLE or lTLE) by targeting Grik2 mRNA and reducing (e.g., knocking down) the expression of GluK2-containing KARs in neurons or astrocytes, which promotes, for example, a reduction in spontaneous epileptiform discharges in neuronal circuits (e.g., hippocampal circuits). Thus, the compositions and methods described herein target the physiological causes of the disease and can be used for treatment.

[0152] Inhibitory polynucleotides targeting Grik2 mRNA Clinical management of TLE is notoriously difficult, with at least one-third of TLE patients unable to adequately control their debilitating seizures using available medications. These patients often experience recurrent epileptic seizures that do not respond to treatment. In such scenarios, TLE patients may resort to invasive and irreversible surgical resection of the epileptogenic focus in the temporal lobe, which can result in undesirable cognitive deficits. Therefore, a significant proportion of TLE patients are in need of novel therapeutic approaches to treat drug-resistant TLE. The compositions and methods described herein offer the advantage of treating the underlying molecular pathophysiology that leads to the onset and progression of TLE.

[0153] The compositions described herein, which are polynucleotides encoding inhibitory nucleic acid constructs (e.g., inhibitory RNA agents or nucleic acid vectors encoding same) targeting Grik2 mRNA (e.g., any one of SEQ ID NOS: 164-174), can be administered according to the methods described herein to treat epilepsy, such as TLE. The methods and compositions described herein can be used to treat TLE patients having any type of TLE, such as TLE with focal seizures, TLE with generalized seizures, mTLE, or lTLE. Furthermore, the methods and compositions of the present disclosure can be used to treat TLE resulting from any etiology, such as mesial temporal sclerosis, traumatic brain injury, brain infection (e.g., encephalitis and meningitis), hypoxic brain injury, stroke, brain tumor, genetic syndrome, or febrile convulsion. The compositions and methods described herein can also be administered as a prophylactic treatment to subjects at risk of developing TLE, e.g., subjects in the latent stage of TLE progression.

[0154] According to the methods and compositions disclosed herein, an inhibitory nucleic acid (e.g., an inhibitory RNA agent) can inhibit expression of GluK2 by causing degradation of Grik2 mRNA in a cell (e.g., a neuron, e.g., a hippocampal neuron, e.g., a hippocampal neuron of the dentate gyrus, e.g., a dentate gyrus granule cell (DGC), or a glutamatergic pyramidal neuron), thereby preventing translation of the mRNA into functional GluK2 protein.

[0155] The inhibitory nucleic acid molecules (e.g., inhibitory RNA agents) targeting Grik2 mRNA disclosed herein can act to reduce the frequency of epileptic brain activity (e.g., epileptiform discharges) in one or more brain regions, or completely inhibit its occurrence. Such brain regions can include, but are not limited to, the medial temporal lobe, lateral temporal lobe, frontal lobe, or more specifically, the hippocampus (e.g., DG, CA1, CA2, CA3, subiculum) or neocortex. Due to the abnormal expression of GluK2-containing KARs in rMF-DGCs of the DG, the occurrence of epileptic brain activity can be inhibited in the DG.

[0156] Accordingly, the present disclosure provides methods and compositions for reducing epileptiform discharges in CNS cells (e.g., DGCs) by contacting the cells with an effective amount of an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to any one of SEQ ID NOs: 1-19, 34-62, 97-108, 133-147, 226-229, and 238-241, or a nucleic acid vector encoding same, such as a nucleic acid vector having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 256. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a miR-30 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:19, a miR-30 stem-loop sequence having at least 85% sequence identity to SEQ ID NO:4, and a miR-30 passenger sequence having at least 85% identity to SEQ ID NO:34. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a miR-30 guide sequence having nucleic acid sequence identity to SEQ ID NO:19, a miR-30 stem-loop sequence having the nucleic acid sequence of SEQ ID NO:4, and a miR-30 passenger sequence having the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', the nucleic acid sequence of SEQ ID NO:4.In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:141, a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:135, and a miR-218-1 passenger sequence having at least 85% sequence identity to SEQ ID NO:147. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a miR-218-1 guide sequence having the nucleic acid sequence of SEQ ID NO: 141, a miR-218-1 stem-loop sequence having the nucleic acid sequence of SEQ ID NO: 135, and a miR-218-1 passenger sequence having the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', the nucleic acid sequence of SEQ ID NO: 135.

[0157] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:19, a miR-30 stem-loop sequence having at least 85% sequence identity to SEQ ID NO:4, and a miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34, and (b) a miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:141. For example, a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity to SEQ ID NO: 135, a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity to SEQ ID NO: 147, and a miR-218-1 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity to SEQ ID NO: 147. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a miR-30 sequence guide sequence having the sequence of SEQ ID NO: 19, a miR-30 stem-loop sequence having the sequence of SEQ ID NO: 4, and a miR-30 passenger sequence having the sequence of SEQ ID NO: 34, and (b) a miR-218-1 guide sequence having the sequence of SEQ ID NO: 141, a miR-218-1 stem-loop sequence having the sequence of SEQ ID NO: 135, and a miR-218-1 passenger sequence having the sequence of SEQ ID NO: 147. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 258. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 258.

[0158] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to any one of SEQ ID NOs: 194-198; (b) an miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 19; an miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 4; and (c) an miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 34. , 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO: 141; and (c) a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135. and a miR-218-1 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147.In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:198; (b) an miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:19; an miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:4; and an miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:34. (c) a miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 141, and (d) a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135. The miR-218-1 stem-loop sequence has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147, and a miR-218-1 passenger sequence has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having the sequence of SEQ ID NO: 198; (b) a miR-30 guide sequence having the sequence of SEQ ID NO: 19, a miR-30 stem-loop sequence having the sequence of SEQ ID NO: 4, and a miR-30 passenger sequence having the sequence of SEQ ID NO: 34; and (c) a miR-218-1 guide sequence having the sequence of SEQ ID NO: 141, a miR-218-1 stem-loop sequence having the sequence of SEQ ID NO: 135, and a miR-218-1 passenger sequence having the sequence of SEQ ID NO: 147.In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 259. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 259.

[0159] In some embodiments of any of the nucleic acid molecules described herein below, the nucleic acid molecule may comprise a single promoter capable of controlling the expression of one or more (e.g., two) miRNA sequences, or two promoters, each capable of controlling the expression of a single miRNA construct. For example, in some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a promoter sequence; (b) a miRNA sequence, such as a miR-30 sequence comprising a miR-30 guide sequence, a miR-30 stem-loop sequence, and a miR-30 passenger sequence; (c) optionally, a second promoter sequence; and (d) a second miRNA sequence, such as a miR-218 sequence comprising a miR-218-1 guide sequence, a miR-218-1 stem-loop sequence, and a miR-218-1 passenger sequence. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a promoter sequence; (b) an miRNA sequence, such as an miR-30 sequence comprising an miR-30 guide sequence, an miR-30 stem-loop sequence, and an miR-30 passenger sequence; and (c) a second miRNA sequence, such as an miR-218 sequence comprising an miR-218-1 guide sequence, an miR-218-1 stem-loop sequence, and an miR-218-1 passenger sequence. In some embodiments, the nucleic acid molecule comprises, from 5' to 3': (a) a promoter sequence; (b) an miRNA sequence, such as an miR-30 sequence comprising an miR-30 guide sequence, an miR-30 stem-loop sequence, and an miR-30 passenger sequence; (c) a second promoter sequence; and (d) a second miRNA sequence, such as an miR-218 sequence comprising an miR-218-1 guide sequence, an miR-218-1 stem-loop sequence, and an miR-218-1 passenger sequence.

[0160] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to any one of SEQ ID NOs: 194-198; (b) a miRNA having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 19; an miR-30 sequence guide sequence, an miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:4, and an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34; (c) an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:141; a miR-218-1 guide sequence having 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135; a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147; and (d) a rabbit beta globin (RBG) adenylation (polyA) signal sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215.In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 198; and (b) a miR-3 promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 19. (c) a miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:4, and a miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34; a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135; a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147; and (d) an RBG polyA signal sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215.In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) an hSyn promoter sequence having the sequence of SEQ ID NO: 198, (b) a miR-30 guide sequence having the sequence of SEQ ID NO: 19, a miR-30 stem-loop sequence having the sequence of SEQ ID NO: 4, and a miR-30 passenger sequence having the sequence of SEQ ID NO: 34, (c) a miR-218-1 guide sequence having the sequence of SEQ ID NO: 141, a miR-218-1 stem-loop sequence having the sequence of SEQ ID NO: 135, and a miR-218-1 passenger sequence having the sequence of SEQ ID NO: 147, and (d) an RBG polyA signal sequence having the sequence of any of SEQ ID NOs: 213, 214, and 215. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 260. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:260.

[0161] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a 5' ITR sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:208; (b) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:198; and (c) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:198. a miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:4; a miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:34; and a miR-30 pathway sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34. (d) a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 141, a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135, and a miR-218-1 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147. 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215; and (f) an RBG polyA signal sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215.In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a 5' ITR sequence having the sequence of SEQ ID NO:208, (b) an hSyn promoter sequence having the sequence of SEQ ID NO:198, (c) an miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:19, an miR-30 stem-loop sequence having the sequence of SEQ ID NO:4, and an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34, and (d) an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:141. (e) a miR-218-1 guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215; (f) a 3' ITR sequence having the sequence of SEQ ID NO: 212; In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 261. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 261.

[0162] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a 5' ITR sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:208; (b) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:198; and (c) an hSyn promoter sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:198. a miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:4; a miR-30 stem-loop sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:34; (d) a miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 141; a miR-218-1 stem-loop having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 135. (e) an miR-218-1 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 147; (e) an RBG polyA signal sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215;(f) a stuffer sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 250 and 251, and (g) a 3' ITR sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO: 212. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', (a) a 5' ITR sequence having the sequence of SEQ ID NO:208; (b) an hSyn promoter sequence having the sequence of SEQ ID NO:198; (c) an miR-30 sequence guide sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to SEQ ID NO:19; an miR-30 stem-loop sequence having the sequence of SEQ ID NO:4; and an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:34; and (d) an miR-30 passenger sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity to SEQ ID NO:141. , 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215; (e) a RBG polyA signal sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 213, 214, and 215; (f) a stuffer sequence having the sequence of one or more (e.g., 2, 3, 4, or 5) of SEQ ID NOs: 250 and 251; and (g) a 3' ITR sequence having the sequence of SEQ ID NO: 212;

[0163] In some embodiments, the nucleic acid molecule is encoded by an expression cassette that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the expression cassette has the nucleic acid sequence of SEQ ID NO: 256.

[0164] The inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) of the present disclosure can be a GluK2 inhibitor. In particular, the GluK2 inhibitor can be a Grik2 mRNA expression inhibitor. Inhibiting the expression of GluK2 can also inhibit the level of GluK5 (Ruiz et al., J Neuroscience 2005). Without wishing to be bound by any theory, the present disclosure is based on the principle that sufficient removal of GluK2 alone should remove all GluK2 / GluK5 heteromers because the GluK5 subunit alone cannot form homomeric assemblies.

[0165] In accordance with the disclosed methods and compositions, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) disclosed herein can have a length of 15 to 50 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, 30, 35, 40, 45, or up to 50 nucleotides). For example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) disclosed herein can have a length of 15 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 16 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 17 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 18 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 19 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 20 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 21 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 22 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 23 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 24 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 25 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 25-30 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 30-35 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 35-40 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 40-45 nucleotides. In another example, an inhibitory nucleic acid molecule (e.g., an inhibitory RNA agent) has a length of 45-50 nucleotides.

[0166] Inhibitory RNA agents of the present disclosure comprise a sequence at least substantially complementary or fully complementary to a region of the sequence of Grik2 mRNA (e.g., any one of SEQ ID NOS: 164-174) or a variant thereof, wherein the complementarity is sufficient to result in specific binding under intracellular conditions. In some embodiments, an inhibitory RNA agent comprises a sequence at least substantially complementary or fully complementary to a region of the sequence of Grik2 mRNA, such as SEQ ID NO: 164, or a variant thereof having at least 85% sequence identity to SEQ ID NO: 164. For example, the present disclosure contemplates inhibitory RNA agents having an antisense sequence complementary to at least seven (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or more) consecutive nucleotides of one or more regions of Grik2 mRNA. In particular examples, an inhibitory RNA agent has an antisense sequence complementary to seven consecutive nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 8 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 9 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 10 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 11 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 12 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 13 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 14 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, an inhibitory RNA agent has an antisense sequence complementary to 15 contiguous nucleotides of one or more regions of Grik2 mRNA.In another example, the inhibitory RNA agent has an antisense sequence complementary to 16 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 17 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 18 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 19 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 20 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 21 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence complementary to 22 contiguous nucleotides of one or more regions of Grik2 mRNA. In yet another example, the inhibitory RNA agent has an antisense sequence that is 100% complementary to the nucleotides of one or more regions of Grik2 mRNA.

[0167] The present disclosure contemplates inhibitory RNA agents that, when bound to one or more regions of Grik2 mRNA (e.g., any one of the regions of Grik2 mRNA set forth in SEQ ID NOs: 164-174), form a duplex structure with Grik2 mRNA that is 7 to 22 (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) nucleotides in length. In some embodiments, an inhibitory RNA agent of the present disclosure can bind to a region of Grik2 mRNA within the sequence of SEQ ID NO: 164 and form a duplex structure with Grik2 mRNA that is 7 to 22 (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) nucleotides in length. For example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 7 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 8 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 9 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 10 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 11 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 12 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 13 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 14 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 15 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 16 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 17 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 18 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 19 nucleotides in length. In another example, the duplex structure between an inhibitory RNA agent and Grik2 mRNA can be 20 nucleotides in length.In another example, the duplex structure between an inhibitory RNA agent and a Grik2 mRNA can be 21 nucleotides in length. In yet another example, the duplex structure between an inhibitory RNA agent and a Grik2 mRNA can be 10 nucleotides in length.

[0168] According to the disclosed methods and compositions, a duplex structure formed by an inhibitory RNA agent (e.g., an agent having at least 85% (at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-19, 34-62, 97-108, 133-147, 226-229, and 238-241), such as an inhibitory RNA agent having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 258, wherein one or more regions of Grik2 mRNA may contain at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) mismatch. For example, the duplex structure may contain one mismatch. In another example, the duplex structure contains two mismatches. In another example, the duplex structure contains three mismatches. In another example, the duplex structure contains four mismatches. In another example, the duplex structure contains five mismatches. In another example, the duplex structure contains six mismatches. In another example, the duplex structure contains seven mismatches. In another example, the duplex structure contains eight mismatches. In another example, the duplex structure contains nine mismatches. In another example, the duplex structure contains ten mismatches. In another example, the duplex structure contains eleven mismatches. In another example, the duplex structure contains twelve mismatches. In another example, the duplex structure contains thirteen mismatches. In another example, the duplex structure contains fourteen mismatches. In yet another example, the duplex structure contains fifteen mismatches.

[0169] Accordingly, the present disclosure relates to isolated, synthetic, or recombinant inhibitory nucleic acid molecules (e.g., inhibitory RNA agents) that target Grik2 mRNA. The inhibitory RNA agents of the present disclosure can be of any suitable type, including RNA or DNA inhibitory polynucleotides. Accordingly, the disclosed methods and compositions feature inhibitors of Grik2 expression that are inhibitory RNA agents (e.g., siRNA, shRNA, miRNA, or shmiRNA). Inhibitory RNA agents, including antisense RNA molecules and antisense DNA molecules, can act to directly block translation of Grik2 mRNA by binding to it and preventing protein translation or by increasing mRNA degradation, thereby reducing GluK2 protein levels and activity. For example, inhibitory RNA agents having at least about 19 bases and complementary to a unique region of the mRNA transcript sequence encoding GluK2 can be synthesized, for example, by conventional techniques (e.g., techniques disclosed herein) and administered, for example, by intravenous injection or infusion, for example, by direct injection into a brain region, among other routes described herein. Methods for using antisense technology to specifically attenuate gene expression of genes whose sequences are known are well known in the art (see, e.g., U.S. Pat. Nos. 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321, and 5,981,732, each of which is incorporated by reference in its entirety).

[0170] In certain examples, the Grik2 inhibitory RNA agent of the present disclosure can be a small interfering RNA (siRNA). Grik2 gene expression can be reduced by contacting a subject or cell with a small double-stranded RNA (dsRNA) or a vector encoding the same, thereby causing the production of small double-stranded RNA that can specifically inhibit Grik2 expression by sequence-specific degradation of mRNA (e.g., via the RNA interference pathway). Methods for selecting an appropriate dsRNA or a vector encoding a dsRNA are known in the art for genes whose sequences are known (see, e.g., Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, G. J. (2002); McManus, M. T. et al. (2002); Brummelkamp, ​​T. R. et al. (2002); U.S. Patent Nos. 6,573,099, 6,506,559; and WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836, each of which is incorporated by reference in its entirety).

[0171] The Grik2 inhibitory RNA agent of the present disclosure can also be a short hairpin RNA (shRNA). shRNA is an RNA sequence that forms a tight hairpin turn and can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into target cells, which often utilizes the ubiquitous U6 promoter to ensure constitutive expression of the shRNA. This vector is usually passed to daughter cells, allowing gene silencing to be maintained after cell division. The shRNA hairpin structure is cleaved into siRNA by the cellular machinery, and the siRNA then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the siRNA sequence to which it is bound.

[0172] Furthermore, the Grik2 expression inhibitor of the present disclosure may be a microRNA (miRNA). miRNA has its general meaning in the art and refers to a microRNA molecule that is generally 21-22 nucleotides in length, although lengths ranging from 19 nucleotides to a maximum of 23 nucleotides have been reported and can be used to suppress the translation of target mRNAs. Each miRNA is processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non-protein-coding genes. Precursor miRNAs have two complementary regions that allow them to form a stem-loop or foldback-like structure that is cleaved in animals by an RNase III-like nuclease enzyme called Dicer. The processed miRNA is typically a portion of the stem that contains a "seed sequence" (typically 6-8 nucleotides) that is fully or substantially complementary to a region of the target mRNA. The processed miRNA (also referred to as "mature miRNA") becomes part of a larger complex for downregulating specific target genes (e.g., reducing translation or degrading the mRNA).

[0173] Furthermore, the GluK2 inhibitors of the present disclosure may be miRNA-compatible shRNAs (shmiRNAs). A shmiRNA agent refers to a chimeric molecule that incorporates an antisense sequence into the -5p or -3p arm of a microRNA scaffold (e.g., an E-miR-30 scaffold) containing the flanking and loop sequences of a microRNA. Compared to shRNAs, shmiRNAs generally have a longer stem-loop structure based on the sequence derived from the microRNA, and the -5p and -3p arms exhibit complete or substantial complementarity (e.g., mismatches, G:U wobble). Due to their longer sequences and processing requirements, shmiRNAs are generally expressed from Pol II promoters. These constructs have also been shown to exhibit reduced toxicity compared to shRNA-based drugs.

[0174] Multiple miRNAs can be used to knock down Grik2 mRNA expression (and subsequently its gene product, GluK2). The miRNAs can be complementary to different target transcripts or different binding sites on a single target transcript. Polycistronic or multigene transcripts can also be utilized to increase the efficiency of target gene knockdown. Multiple genes encoding the same or different miRNAs can be regulated together in a single transcript or as separate transcripts in a single vector cassette. The miRNAs of the present disclosure can be packaged into vectors, such as viral vectors (including, but not limited to, recombinant adeno-associated virus (rAAV) vectors, lentiviral vectors, retroviral vectors, and retrotransposon-based vector systems).

[0175] Inhibitory RNAs complementary to (e.g., substantially or completely complementary to) the sense target sequence of Grik2 mRNA are generally encoded by DNA sequences for the production of any of the aforementioned inhibitors (e.g., siRNA, shRNA, miRNA, or shmiRNA). DNA encoding the desired double-stranded RNA can be incorporated into a gene cassette (e.g., an expression cassette in which transcription of the DNA is controlled by a promoter). Improved RISC loading of guide sequences

[0176] A step in RNA interference is the assembly of a microRNA guide strand into the RNA-induced silencing complex (RISC) protein complex, which mediates target mRNA cleavage. MicroRNAs are produced as double-stranded duplexes containing a guide strand hybridized to a passenger strand through complementary base pairing. Assembly of the guide strand into the RISC complex is generally accompanied by degradation of the passenger strand. RISC assembly favors microRNA strands with 5' ends that are more prone to fraying or breaking free from the duplex. The constructs described herein are designed to favor guide selection and loading and disfavor passenger selection by RISC by destabilizing base pairing at the 5' end of the guide strand (e.g., by introducing a UA or UG wobble pair at or near the 5' end of the guide strand) and strengthening base pairing at the 5' end of the passenger strand (e.g., by introducing a GC pair at or near the 5' end of the passenger strand). This strategy is achievable because mismatches between the guide strand and the target mRNA are well tolerated if they occur at the first nucleotide or near the 3' end of the guide strand (e.g., within the last four nucleotides). Such a strategy not only improves on-target knockdown by the guide strand, but also reduces off-target effects from the production or retention of the passenger strand by the RISC protein complex.

[0177] Thus, the anti-Grik2 antisense molecules (e.g., microRNA, shRNA, siRNA, or shmiRNA) described herein contain one or more modifications that improve RISC loading or retention of the guide strand, decrease RISC loading or retention of the passenger strand, increase the guide to passenger strand ratio in the cell, and increase the level of knockdown of the target Grik2 mRNA.

[0178] Base pairing instability at or near the 5' end of the guide strand has been increased in some of the constructs described herein to improve RISC loading or retention of the guide strand. For example, base pairing instability is achieved by introducing a UA pair or a UG wobble pair at or near the 5' end of the guide strand in some constructs.

[0179] Some of the constructs described herein reduce RISC loading or retention of the passenger strand by introducing base-pairing instability at the 5' end of the passenger strand by adding a CG pair at or near the 5' end of the passenger strand.

[0180] Some constructs of the present disclosure were also designed to enhance RISC loading or retention of the guide strand by introducing a 5'-terminal uracil into the guide strand, a 5'-terminal nucleotide that does not participate in hybridization to the target mRNA (e.g., Grik2 mRNA) and is generally anchored in the phosphate-binding pocket of the Argonaute RISC catalytic component 2 (Ago2) protein.

[0181] For some of the disclosed constructs, introducing one or more mismatches (e.g., 1, 2, 3, 4, 5, 6, 7, or more mismatches) into the seed region of the guide strand (corresponding to nucleotides 2-7 of the guide strand; g2-g7) reduces RISC loading or retention of the passenger strand. This strategy is used to facilitate unwinding and removal of the passenger strand during RISC loading. Extensive complementarity in the seed region (guide nucleotides 2-8, g2-g8) and the middle region of the guide strand is crucial for Ago2-mediated mRNA cleavage, but base pairing at the 3' end is not required. Indeed, mismatches with the target mRNA at positions g18, g19, g20, and g21 of the guide strand were determined to attenuate guide strand release from Ago2, i.e., target mRNA-mediated unloading activity.

[0182] Dicer cleavage of the loop region from the stem-loop structure of anti-Grik2 constructs is improved for some of the constructs disclosed herein by strengthening base pairing at the junction between the stem and loop regions by replacing a UG wobble pair with a CG pair. The Grik2 mRNA-targeting constructs of the present disclosure utilize the aforementioned modifications to promote an increased ratio of guide to passenger strand production, improving silencing of Grik2 for the treatment of seizure disorders (e.g., TLE).

[0183] Thus, the inhibitory RNA molecules described herein can comprise stem-loop sequences containing rationally designed guide and passenger strand sequences from the anti-Grik2 sequence GI (SEQ ID NO: 16) and complementary sequences thereto (see, e.g., Table 2 (e.g., SEQ ID NOs: 1-15, 226-229, and 238-241)) incorporated into the E-miR-30 microRNA scaffold, or variants thereof having at least 85% sequence identity thereto (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0184] Thus, Grik2-targeting antisense constructs of the present disclosure may comprise guide strand (SEQ ID NOs: 16-30, 230-233, and 242-245) and passenger strand (SEQ ID NOs: 31-45, 234-237, and 246-249) pairs as set forth in Table 3 below. [Table 3-1] [Table 3-2]

[0185] Also disclosed herein are inhibitory RNA molecules that may include stem-loop sequences containing rationally designed guide and passenger strand sequences from the anti-Grik2 sequence G9 (SEQ ID NO: 63) and complementary sequences thereto (see, e.g., Table 4 (e.g., SEQ ID NOs: 46-62)) incorporated into an E-miR-124-3 microRNA scaffold, or variants thereof having at least 85% sequence identity thereto (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0186] Thus, Grik2-targeting antisense constructs of the present disclosure may comprise guide strand (SEQ ID NOs: 63-79) and passenger strand (SEQ ID NOs: 80-96) pairs set forth in Table 5 below. [Table 5-1] [Table 5-2]

[0187] The inhibitory RNA molecules described herein may comprise stem-loop sequences comprising rationally designed guide and passenger strand sequences from the anti-Grik2 sequence MW (SEQ ID NO: 109) and complementary sequences thereto (see, e.g., Table 6 (e.g., SEQ ID NOs: 97-108)) incorporated into the E-miR-124-3 microRNA scaffold, or variants thereof having at least 85% sequence identity thereto (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)). [Table 6-1] [Table 6-2] [Table 6-3]

[0188] Thus, Grik2-targeting antisense constructs of the present disclosure may comprise guide strand (SEQ ID NOs: 109-120) and passenger strand (SEQ ID NOs: 121-132) pairs set forth in Table 7 below. [Table 7-1] [Table 7-2]

[0189] The inhibitory RNA molecules described herein may comprise stem-loop sequences comprising rationally designed guide and passenger strand sequences from the anti-Grik2 sequence MW (SEQ ID NO: 109) and complementary sequences thereto (see, e.g., Table 8 (e.g., SEQ ID NOs: 133-138)) incorporated into the E-miR-218 microRNA scaffold, or variants thereof having at least 85% sequence identity thereto (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)). [Table 8]

[0190] Thus, Grik2-targeting antisense constructs of the present disclosure may comprise guide strand (SEQ ID NOs: 139-144) and passenger strand (SEQ ID NOs: 145-146) pairs set forth in Table 9 below. [Table 9]

[0191] The aforementioned sequences are represented as DNA (i.e., cDNA) sequences that can be incorporated into the vectors of the present disclosure. These sequences can also be represented as corresponding RNA sequences synthesized from the vectors in cells. Those skilled in the art will understand that cDNA sequences are equivalent to mRNA sequences, except that uridines are replaced with thymidines, and can be used for the same purpose as described herein, i.e., to generate polynucleotides for inhibiting the expression of Grik2 mRNA. In the case of DNA vectors (e.g., AAV), the polynucleotide containing the antisense nucleic acid is a DNA sequence. In the case of RNA vectors, the transgene cassette incorporates the RNA equivalent of the antisense DNA sequence described herein.

[0192] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:1. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:1. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:1. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:1.

[0193] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:2. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:2.

[0194] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:3. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:3. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:3. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:3.

[0195] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:4. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:4.

[0196] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:5. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:5. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:5. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:5.

[0197] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:6. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:6. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:6. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:6.

[0198] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:7. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:7. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:7. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:7.

[0199] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:8. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:8. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:8. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:8.

[0200] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:9. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:9. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:9. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:9.

[0201] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 10. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 10. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 10. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 10.

[0202] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:11. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:11. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:11. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:11.

[0203] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 12. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 12.

[0204] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 12. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 13.

[0205] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 14. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 14.

[0206] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 15. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 15.

[0207] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:226. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:226. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:226. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:226.

[0208] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:227. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:227. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:227. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:227.

[0209] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:228. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:228. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:228. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:228.

[0210] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:229. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:229. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:229. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:229.

[0211] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:238. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:238. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:238. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:238.

[0212] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:239. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:239. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:239. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:239.

[0213] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:240. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:240. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:240. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:240.

[0214] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:241. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:241. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:241. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:241.

[0215] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 46. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 46. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 46. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 46.

[0216] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 47. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 47. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 47. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 47.

[0217] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 48. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 48. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO: 48. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 48.

[0218] An inhibitory RNA sequence of the present disclosure can have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:49. For example, an inhibitory RNA can have at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:49. In another example, an inhibitory RNA can have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to the nucleic acid sequence of SEQ ID NO:49. In a further example, an inhibitory RNA can have the nucleic acid sequence of SEQ ID NO:49.

[0219] An inhibitory RNA sequence of the present...

Claims

1. 1. An isolated polynucleotide that specifically binds to Grik2 mRNA comprising a stem-loop region comprising a 5' arm (5p), a loop region, and a 3' arm (3p), wherein the stem-loop region comprises a guide strand sequence and a passenger strand sequence, and the guide strand sequence and the passenger strand sequence are (a) a uracil (U)-adenine (A) base pair or a U-guanine (G) base pair at the 5' end of the guide strand; (b) a cytosine (C)-G pair at the 5' end of the passenger strand; (c) a U at the 5' end of the guide strand sequence; (d) a mismatch in the seed region between the guide strand sequence and the passenger strand sequence; and / or (e) comprising a C-G base pair or a U-A base pair to replace a U-G wobble at the junction of the stem and loop regions of the polynucleotide. An isolated polynucleotide.

2. (i) (a) and (c) improve guide strand sequence loading into an RNA-induced silencing complex (RISC) protein. (ii)(b) impairs passenger strand sequence loading into the RISC protein; (iii)(d) promotes decoupling of the passenger strand sequence from the guide strand sequence during RISC loading; (iv) (e) improves cleavage of the loop region from the stem region by Dicer; and / or (v) the seed region of the guide strand sequence comprises nucleotides 2 to 7 of the guide strand sequence. (i) the stem-loop region is a polynucleotide having at least 85% sequence identity to a nucleic acid sequence of SEQ ID NO:4 or an RNA thereof, the guide strand sequence has a nucleic acid sequence of SEQ ID NO:19 or an RNA thereof, and / or the passenger strand sequence has a nucleic acid sequence of SEQ ID NO:34 or an RNA thereof. (ii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 135 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 141 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 147 or its RNA; (iii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 2 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 17 or its RNA, and the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 32 or its RNA; (iv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 3 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 18 or its RNA, and the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 33 or its RNA; (v) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:5 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:20 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:35 or its RNA; (vi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 6 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 21 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 36 or its RNA; (vii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 22 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 37 or its RNA; (viii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 8 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 23 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 38 or its RNA; (ix) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 9 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 24 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 39 or its RNA; (x) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 10 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 25 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 40 or its RNA; (xi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 11 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 26 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 41 or its RNA. (xii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 12 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 27 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 42 or its RNA; (xiii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 28 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 43 or its RNA; (xiv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 29 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 44 or its RNA. (xv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 30 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 45 or its RNA. (xvi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 226 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 230 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 234 or its RNA; (xvii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 227 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 231 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 235 or its RNA; (xviii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 228 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 232 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 236 or its RNA; (xix) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 229 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 233 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 237 or its RNA. (xx) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 238 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 242 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 246 or its RNA; (xxi) The stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 239 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 243 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 247 or its RNA. (xxii) The stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 240 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 244 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 248 or its RNA. (xxiii) The stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 241 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 245 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 249 or its RNA. (xxiv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 47 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 64 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 81 or its RNA. (xxv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 48 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 65 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 82 or its RNA. (xxvi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 49 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 66 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 83 or its RNA. (xxvii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:50 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:67 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:84 or its RNA. (xxviii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:51 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:68 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:85 or its RNA. (xxix) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:52 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:69 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:86 or its RNA. (xxx) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:53 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:70 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:87 or its RNA. (xxxi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:54 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:71 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:88 or its RNA. (xxxii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:55 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:72 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:89 or its RNA. (xxxiii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:56 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:73 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:90 or its RNA. (xxxiv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:57 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:74 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:91 or its RNA. (xxxv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:58 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:75 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:92 or its RNA. (xxxvi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:59 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO:76 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO:93 or its RNA. (xxxvii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 60 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 77 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 94 or its RNA. (xxxviii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 61 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 78 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 95 or its RNA. (xxxix) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 62 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 79 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 96 or its RNA. (xl) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 98 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 110 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 122 or its RNA; (xli) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 99 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 111 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 123 or its RNA; (xlii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 100 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 112 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 124 or its RNA. (xliii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 101 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 113 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 125 or its RNA. (xliv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 102 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 114 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 126 or its RNA; (xlv) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 103 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 115 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 127 or its RNA; (xlvi) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 104 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 116 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 128 or its RNA; (xlvii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 105 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 117 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 129 or its RNA; (xlviii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 106 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 118 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 130 or its RNA; (xlix) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 107 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 119 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 131 or its RNA; (l) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 108 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 120 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 132 or its RNA; (li) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 134 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 140 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 146 or its RNA; (lii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 136 or its RNA, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 142 or its RNA, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 148 or its RNA; (liii) the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 137 or RNA thereof, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 143 or RNA thereof, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 149 or RNA thereof; or (liv) The polynucleotide of claim 1, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 138 or RNA thereof, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 144 or RNA thereof, and / or the passenger strand sequence has the nucleic acid sequence of SEQ ID NO: 150 or RNA thereof.

4. The polynucleotide of claim 1 , wherein the polynucleotide comprises an antisense oligonucleotide (ASO).

5. 5. The polynucleotide of claim 4, wherein the polynucleotide comprises a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA) or a short hairpin-compatible miRNA (shmiRNA).

6. The polynucleotide of claim 1 , wherein the polynucleotide is 19, 20, or 21 nucleotides. (i) the Grik2 mRNA is encoded by the nucleic acid sequence of SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173 or SEQ ID NO:174; and / or (ii) The polynucleotide of claim 1, wherein the polynucleotide is capable of reducing the level of GluK2 protein in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

8. The polynucleotide of claim 7 , wherein the cell is a human cell and / or a neuron, and the neuron is a hippocampal neuron.

9. The polynucleotide of claim 8, wherein the hippocampal neuron is a dentate gyrus granule cell (DGC) or a glutamatergic pyramidal neuron.

10. A vector comprising the polynucleotide according to any one of claims 1 to 9, said vector comprising: (i) replication-deficient, (ii) an expression vector, and / or (iii) a mammalian, insect, bacterial or viral vector That is, a vector.

11. An expression cassette comprising: I) a polynucleotide comprising a stem-loop sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 4, 135, 256, 1-3, 5-15, 46-62, 97-108, 133, 134, 136-138, 226-229, 238-241, and 257-261; and II) (a) a 5' flanking region comprising a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 217, 220, or 223; (b) a 3' flanking region comprising a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 218, 221, or 224; and / or (c) a loop region comprising a microRNA loop sequence that is an E-miR-30, miR-218-1, or E-miR-124-3 sequence, wherein the microRNA loop sequence comprises a polynucleotide having at least 85% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 219, 222, or 225. An expression cassette comprising:

12. An expression cassette comprising, from 5' to 3': (a) a promoter sequence; (b) a first polynucleotide comprising a stem-loop sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 4, 19, 34, 135, 141, 147, 1-3, 5-18, 35-62, 97-108, 133, 134, 136-140, 142-146, 226-229, or 238-241; (c) a second polynucleotide comprising a stem-loop sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 4, 19, 34, 135, 141, 147, 1-3, 5-18, 35-62, 97-108, 133, 134, 136-140, 142-146, 226-229, or 238-241; Expression cassette.

13. The first polynucleotide and the second polynucleotide each independently comprise a passenger sequence complementary or substantially complementary to a guide sequence, the passenger sequence being located 5' or 3' to the guide sequence, and the expression cassette comprises: (i) a 5' flanking region located 5' to the guide sequence; (ii) a 3' flanking region located 3' to the guide sequence; and (iii) a loop region located between the guide sequence and the passenger sequence.

13. The expression cassette of claim 12, further comprising: wherein the loop region comprises a microRNA loop sequence, and the microRNA loop sequence is an E-miR-30, miR-218-1, or E-miR-124-3 sequence.

14. (i) the 5' flanking region comprises a polynucleotide having at least 85% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 217, 220, or 223; (ii) the 3' flanking region comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 218, 221, or 224; and (iii) the microRNA loop sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 219, 222, or 225. (i) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the expression cassette; (ii) a 3'-ITR sequence at the 3' end of the expression cassette; (iii) an enhancer sequence, (iv) intron sequences, (v) one or more polyadenylation signal sequences, wherein the one or more polyadenylation signal sequences are the rabbit beta globin (RBG) polyadenylation signal sequence or the bovine growth hormone (BGH) polyadenylation signal sequence, and / or (vi) a promoter selected from the group consisting of hSyn promoter and CaMKII promoter. The expression cassette of any one of claims 12 to 14, further comprising: (i) the 5'-ITR sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:208 or SEQ ID NO:209; (ii) the 3'-ITR sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 212, 210, or 211; (iii) the enhancer sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 207; (iv) the intron sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 205 or SEQ ID NO: 206; and / or (v) the RBG polyadenylation signal sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 215, 213, or 214, or the BGH polyadenylation signal sequence comprises a polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:

216.

17. The expression cassette according to claim 11 or 12, wherein the expression cassette is incorporated into a vector.

18. 13. The expression cassette of claim 11 or 12, wherein the expression cassette comprises at least 80% sequence identity to the sequence of SEQ ID NO:

256.

19. 19. The expression cassette of claim 18, wherein the expression cassette comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO:

256.

20. An in vitro method for inhibiting Grik2 expression in a cell, comprising contacting said cell with at least one polynucleotide according to any one of claims 1 to 9, an expression cassette according to claim 11 or 12, or a vector comprising said at least one polynucleotide or said expression cassette.

21. 21. The method of claim 20, wherein the polynucleotide specifically hybridizes to Grik2 mRNA and inhibits or reduces expression of Grik2 in the cell, and the method reduces the level of GluK2 protein in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

22. 22. The method of claim 21 , wherein the cell is a human cell and / or a neuron, and the neuron is a hippocampal neuron.

23. 23. The method of claim 22, wherein the hippocampal neuron is a DGC or a glutamatergic pyramidal neuron.

24. 13. A composition comprising a polynucleotide according to any one of claims 1 to 9, an expression cassette according to claim 11 or 12, or a vector comprising said polynucleotide or said expression cassette, for use in a method for treating or ameliorating a disease or disorder in a subject in need thereof.

25. 25. The composition of claim 24, wherein the disorder is epilepsy.

26. 26. The composition of claim 25, wherein the epilepsy is temporal lobe epilepsy (TLE), chronic epilepsy, and / or refractory epilepsy.

27. 27. The composition of claim 26, wherein the TLE is a lateral TLE (lTLE) or a medial TLE (mTLE).

28. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 9, an expression cassette according to claim 11 or 12, or a vector comprising any one of said polynucleotides or said expression cassettes, and a pharma- ceutically acceptable carrier, diluent or excipient.

29. 30. A kit comprising the pharmaceutical composition of claim 28 and a package insert.

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