TW202346585A - Methods and compositions for treating epilepsy - Google Patents

Abstract

Disclosed are methods and compositions relating to antisense therapy for treating epilepsy, such as a 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 neuronal circuits.

Description

Methods and compositions for treating epilepsy

This 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.

Globally, an estimated 5 million people are diagnosed with epilepsy each year, a neurological disorder characterized by seizures, or sudden and recurring episodes of sensory impairment, loss of consciousness, or convulsions associated with abnormal electrical activity in the brain. A typical diagnosis of epilepsy is made when a patient experiences two or more unprovoked seizures. Causes of epilepsy include genetic abnormalities, previous brain infections, prenatal injuries, developmental disabilities and other neurological problems such as stroke or brain tumors, but approximately 50% of people diagnosed with epilepsy have no known reason for the development of the disease.

Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy in adults (30% to 40% of all forms of epilepsy). The hippocampus is known to play a key role in the pathophysiology of TLE. Abnormal rewiring of neural circuits occurs in human patients and animal models of TLE. One of the best examples of network reorganization ("reactive plasticity") is the sprouting of recurrent mossy fibers (rMF), which establishes novel pathophysiology in the dentate granule cells (DGCs) of the hippocampus. Glutamatergic synapses (Tauck and Nadler, 1985; Represa et al., 1989a, 1989b; Sutula et al., 1989; Gabriel et al., 2004), which lead to recurrent excitatory circuits. rMF synapses act through ectopic kainate receptors (KARs) (Epsztein et al., 2005; Artinian et al., 2011, 2015). KAR is a tetrameric glutamate receptor assembled from GluK1-GluK5 subunits. In heterologous expression systems, GluK1, GluK2, and GluK3 form homologous receptors, while GluK4 and GluK5 form heterologous receptors together with the GluK1 to GluK3 subunits. Natural KARs are widely distributed in the brain, with high densities of receptors found in the hippocampus (Carta et al., 2016, EJN), a key structure involved in TLE. Previous studies by the inventors have determined that epileptic activity, including interictal spikes and ictal discharges, is significantly reduced in mice lacking the GluK2 KAR subunit. Furthermore, epileptiform activity was greatly reduced following the use of pharmacological small molecule antagonists of GluK2/GluK5-containing KARs that block ectopic synaptic KARs (Peret et al., 2014). These data support the hypothesis that KAR manifesting ectopically at rMF in DGCs plays a major role in the chronic onset of TLE. Therefore, aberrant KARs expressed in DGCs and composed of GluK2/GluK5 are considered promising targets for the treatment of drug-resistant epilepsy such as TLE.

RNA interference (RNAi) strategies have been proposed against many disease targets. The successful application of RNAi-based therapies has been limited. RNAi therapies face several challenges, such as the need for repeated dosing and formulation challenges. However, the availability of RNAi-based gene therapies for the treatment of refractory TLE is limited. Therefore, new therapeutic modalities are urgently needed to treat epilepsy, such as TLE (eg, refractory TLE).

The present disclosure provides compositions and methods for treating or preventing epilepsy, such as temporal lobe epilepsy (TLE), in an individual (eg, a human) in need thereof. The disclosed methods include administering to an individual diagnosed with epilepsy or at risk of developing epilepsy a therapeutically effective amount of a polynucleotide (e.g., inhibitory polynucleotide), such as an antisense oligonucleotide (ASO). ), shRNA, siRNA, microRNA or shmiRNA, which targets the mRNA encoded by the glutamate ionotropic receptor kainate subunit 2 ( Grik2 ) gene, or administers a therapeutically effective amount of the encoded Nucleic acid vectors of the polynucleotide (eg, lentiviral vectors or adeno-associated virus (AAV) vectors, eg, AAV9 vectors). 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 comprising one or more of the disclosed inhibitory nucleic acid (eg, RNA) agents and a nucleic acid vector encoding the inhibitory nucleic acid.

This disclosure is based in part on the surprising finding that inhibitory polynucleotides described herein exhibit significantly higher leader to follower ratios (G/P ratios), supporting direct processing of inhibitory polynucleotides. , a substantial increase and subsequent improvement in both the expression level of Grik2 mRNA and the resulting expression level of GluK2 protein. One challenge of microRNA (miRNA) therapy is the inefficiency of processing of transfected polynucleotides. Therefore, an improvement in the G/P ratio may be associated with an increase in the production of mature miRNA molecules and, concomitantly, with an increase in the desired therapeutic effect of the administered miRNA therapy.

In a first aspect, the present disclosure features one or more isolated inhibitory polynucleotides that specifically hybridize to Grik2 mRNA, including a stem-loop region (including the 5' arm (5p), the loop region, the 3 ' arm (3p)), wherein the stem-loop region includes a leader strand sequence and a follower strand sequence, and the leader strand sequence and follower strand sequence include: (a) Uracil (U)-adenine at the 5' end of the leader strand (A) base pair or U-guanine (G) base pair, (b) cytosine (C)-G base pair at the 5' end of the follower strand, (c) at the 5' end of the leader strand sequence U, (d) a mismatch in the seed region between the leader strand sequence and the follower strand sequence; and/or (e) in the stem region and the loop region of the polynucleotide The junction is replaced by a CG base pair or a UA base pair that replaces the UG wobble.

In some embodiments, a) and c) improve a guide strand sequence loaded into an RNA-induced silencing complex (RISC) protein. In some embodiments, b) impair the loading of the follower strand sequence into the RISC protein. In some embodiments, d) facilitates decoupling of the follower strand sequence from the leader strand sequence during RISC loading. In some embodiments, e) enhancing the loop region by cleavage of the stem region by Dicer. In some embodiments, the seed region of the leader sequence includes nucleotides 2 to 7 of the leader sequence.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 2 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the leader of SEQ ID NO: 17 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 17 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 32 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 3 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the leader 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 18 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 33 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 4 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the leader of SEQ ID NO: 19 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 19 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 34 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 5 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the leader of SEQ ID NO: 20 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 20 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 35 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 6 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the leader of SEQ ID NO: 21 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 21 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 36 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 7 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the leader of SEQ ID NO: 22 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 22 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 37 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 8 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the leader of SEQ ID NO: 23 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 23 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 38 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 9 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the leader of SEQ ID NO: 23 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 23 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 39 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 10 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the leader of SEQ ID NO: 25 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 25 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 40 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 11 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 11. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the leader of SEQ ID NO: 4 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 26 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 41 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 12 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 12. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the leader of SEQ ID NO: 27 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 27 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 42 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 13 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 13. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the leader 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 28 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 43. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 43 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 14 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 14. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the leader of SEQ ID NO: 29 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 29 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 44 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 15 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence 15 identity of a polynucleotide. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the leader of SEQ ID NO: 30 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 30 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 45 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 226 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 226. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 230. In some embodiments, the leader of SEQ ID NO: 230 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 230 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 234. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 234 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 227 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 227. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 231. In some embodiments, the leader of SEQ ID NO: 231 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 231 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 235. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 235 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 228 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 228. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 232. In some embodiments, the leader of SEQ ID NO: 232 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 232 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 236. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 236 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 229 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 229. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 233. In some embodiments, the leader of SEQ ID NO: 233 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 233 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 237. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bolded nucleotides in SEQ ID NO: 237 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 238 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 238. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 242. In some embodiments, the leader of SEQ ID NO: 242 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 242 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 246. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 246 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 239 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 239. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 243. In some embodiments, the leader of SEQ ID NO: 243 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 243 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 247. In some embodiments, the companion 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 247 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 240 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 240. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 244. In some embodiments, the leader of SEQ ID NO: 244 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 244 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 248. In some embodiments, the companion 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 248 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 241 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 241. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 245. In some embodiments, the leader of SEQ ID NO: 245 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 245 shown in Table 3. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 249. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 249 shown in Table 3.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 47 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 47. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the leader of SEQ ID NO: 64 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 64 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 81 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 48 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 48. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the leader of SEQ ID NO: 65 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 65 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 82 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 49 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 49. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the leader of SEQ ID NO: 66 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 66 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 83 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 50 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 50. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 67. In some embodiments, the leader of SEQ ID NO: 67 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 67 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 84. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 84 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 51 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 51. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the leader 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 68 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 85. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 85 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 52 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 52. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 69. In some embodiments, the leader of SEQ ID NO: 69 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 69 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 86 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 53 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 53. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 70. In some embodiments, the leader of SEQ ID NO: 70 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 70 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 87. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 87 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 54 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 54. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the leader of SEQ ID NO: 71 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 71 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 88. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 88 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 55 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 55. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 72. In some embodiments, the leader of SEQ ID NO: 72 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 72 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 89. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 89 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 56 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 56. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 73. In some embodiments, the leader of SEQ ID NO: 73 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 73 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 90. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 90 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 57 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 57. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 74. In some embodiments, the leader of SEQ ID NO: 74 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 74 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 91. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 91 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 58 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 58. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, the leader of SEQ ID NO: 75 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 75 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 92. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bolded nucleotides in SEQ ID NO: 92 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 59 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 59. In some embodiments, the leader sequence has SEQ ID NO: 76. In some embodiments, the leader of SEQ ID NO: 76 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 76 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 93. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 93 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 60 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 60. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, the leader of SEQ ID NO: 77 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 77 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 94 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 61 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 61. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 78. In some embodiments, the leader of SEQ ID NO: 78 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 78 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 95. In some embodiments, a follower 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 95 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 62 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 62. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, the leader of SEQ ID NO: 79 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 79 shown in Table 5. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 96. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 96 shown in Table 5.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 98 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 98. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 110. In some embodiments, the leader of SEQ ID NO: 110 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 110 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 122 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 99 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 99. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 111. In some embodiments, the leader of SEQ ID NO: 111 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 111 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 123. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 123 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 100 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 100. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 112. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 112 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 124. In some embodiments, the slave 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 124 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 101 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) the nucleic acid sequence of sequence 101. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 113. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 113 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 125 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 102 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 102. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 114. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 114 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 126. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 126 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 103 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence 103 identity of a polynucleotide. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 115. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 115 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 127. In some embodiments, the companion 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 127 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 104 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence 104 identity of a polynucleotide. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 116. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 116 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 128. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 128 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 105 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 105. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 117. In some embodiments, the companion 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 mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 117 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 129. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 129 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 106 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 106. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 118. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 118 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 130. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 130 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 107 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 107. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 119. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 119 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 131. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 131 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 108 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 108. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 120. In some embodiments, the slave 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 120 shown in Table 7. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 132. In some embodiments, the companion 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 mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 132 shown in Table 7.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 134 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 134. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 140. In some embodiments, the leader of SEQ ID NO: 140 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 140 shown in Table 9. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 146. In some embodiments, the leader of SEQ ID NO: 146 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 146 shown in Table 9.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 135 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 135. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 141. In some embodiments, the leader of SEQ ID NO: 141 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 141 shown in Table 9. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the leader of SEQ ID NO: 147 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 147 shown in Table 9.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 136 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 136. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 142. In some embodiments, the leader of SEQ ID NO: 142 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 142 shown in Table 9. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 148. In some embodiments, the leader of SEQ ID NO: 148 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 148 shown in Table 9.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 137 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 137. In some embodiments, the guide sequence has the nucleic acid sequence of SEQ ID NO: 143. In some embodiments, the leader of SEQ ID NO: 143 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 143 shown in Table 9. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 149. In some embodiments, the leader of SEQ ID NO: 149 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), wherein the change does not involve any of the bold nucleotides in SEQ ID NO: 149 shown in Table 9.

In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 138 , 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the nucleic acid sequence of sequence 138. In some embodiments, the guide strand sequence has the nucleic acid sequence of SEQ ID NO: 144. In some embodiments, the leader of SEQ ID NO: 144 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 144 shown in Table 9. In some embodiments, the companion strand sequence has the nucleic acid sequence of SEQ ID NO: 150. In some embodiments, the leader of SEQ ID NO: 150 includes 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotide changes (e.g., substitutions, deletions, insertions, or mismatch), where the change does not involve any of the bold nucleotides in SEQ ID NO: 150 shown in Table 9.

In some embodiments, the inhibitory polynucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the inhibitory polynucleotide comprises short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or short hairpin-mediated miRNA (shmiRNA).

In some embodiments, the polynucleotide is between 19 and 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.

In some embodiments, the Grik2 mRNA is encoded by a nucleic acid sequence that is 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: 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, the Grik2 mRNA is encoded by the following nucleic acid sequences: 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, the inhibitory polynucleotide is capable of reducing the levels of GluK2 protein in the 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 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 cells are neurons, such as hippocampal neurons (e.g., dentate granule cells (DGCs) or glutamatergic pyramidal neurons). In other embodiments, including those wherein the cell is a neuron, the cell is a human cell.

In another aspect, the present disclosure features a vector comprising a polynucleotide of any of the preceding aspects and embodiments. In some embodiments, the vector is replication defective. 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 system is selected from the group consisting of: adeno-associated virus (AAV), retrovirus, adenovirus, parvovirus, coronavirus, negative-strand RNA virus, orthomyxovirus, rod-shaped virus Viruses, paramyxoviruses, orthostranded RNA viruses, picornaviruses, alphaviruses, double-stranded DNA viruses, herpesviruses, Epstein-Barr virus, cytomegalovirus, fowlpox virus, and canaries Poxvirus. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is an AAV5, AAV9 or AAVrh10 vector.

In another aspect, the disclosure features a representation cassette comprising a polynucleotide encoding or comprising a polynucleotide corresponding to a stem-loop sequence of the first aspect of the disclosure, such as as described below Any of the nucleic acid sequences 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%)) stem-loop regions of sequence identity: SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 226 to SEQ ID NO: 229, and SEQ ID NO: 238 to SEQ ID NO: 241. In some embodiments, the stem-loop region is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 4 , 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the stem-loop region has the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 135 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 258 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 259 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 260 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 261 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 256 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the performance cassette comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 257 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity.

In another aspect, the present disclosure provides a representation cassette comprising a polynucleotide that shares at least 85% (e.g., at least 86%, 87%, 88%) a nucleic acid sequence with any of the following , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO :46-62.

In another aspect, the present disclosure provides a representation cassette comprising a polynucleotide that shares at least 85% (e.g., at least 86%, 87%, 88%) a nucleic acid sequence with any of the following , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO :97-108.

In another aspect, the present disclosure provides a representation cassette comprising a polynucleotide that shares at least 85% (e.g., at least 86%, 87%, 88%) a nucleic acid sequence with any of the following , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO : 133-138. In some embodiments, the stem-loop sequence is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) identical to the nucleic acid sequence of SEQ ID NO: 135 , 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the stem-loop sequence has the nucleic acid sequence of SEQ ID NO: 135.

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

In some embodiments, the expression cassette comprises a synaptophysin (hSyn) promoter or a calcium/calcin-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), U6, H1, or 7SK promoter.

In another aspect, the present disclosure provides a performance cassette comprising, from 5' to 3': (a) a first promoter sequence; (b) a polynucleotide comprising a stem-loop sequence, the stem-loop sequence A nucleic acid sequence that is at least 85% identical to any of the following (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: SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO : 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to SEQ ID NO: 229 or SEQ ID NO: 238 to SEQ ID NO: 241; (c) as appropriate , a second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence that is at least 85% (e.g., at least 86%, 87%, 88%) identical to the nucleic acid sequence of any of the following , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO : SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO: 97 to 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to SEQ ID NO: 229, or SEQ ID NO: 238 to SEQ ID NO: 241. In some embodiments, the performance cassette comprises, from 5' to 3', (a) a first promoter sequence; (b) a polynucleotide comprising a stem-loop sequence identical to SEQ ID NO: 4 The nucleic acid sequence 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; (c) optionally, a second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence identical to SEQ ID NO: 135 nucleic acid sequences have 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. In some embodiments, the performance cassette comprises, from 5' to 3', (a) a first promoter sequence; (b) a polynucleotide comprising a stem-loop sequence having SEQ ID NO: 4 the nucleic acid sequence; (c) optionally, the second promoter sequence; and (d) a polynucleotide comprising a stem-loop sequence, the polynucleotide having the nucleic acid sequence of SEQ ID NO: 135. In some embodiments, the performance cassette includes a sequence that is at least 85% identical 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%)) sequence identity.

In some embodiments, the polynucleotide comprising a stem-loop sequence having SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO: 97 to the nucleic acid sequence of any one of SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to 229, or SEQ ID NO: 238 to SEQ ID NO: 241, or a variant thereof variant, the variant is 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) contains a follower sequence that is complementary or substantially complementary to the leader sequence, wherein the follower sequence is located 5' relative to the leader sequence or 3'. In some embodiments, the polynucleotide comprising a stem-loop sequence having SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO: 97 to the nucleic acid sequence of any one of SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to 229, or SEQ ID NO: 238 to SEQ ID NO: 241, or a variant thereof variant, the variant is 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) contains a 5' flanking region located 5' relative to the leader sequence. In some embodiments, the polynucleotide comprising a stem-loop sequence having SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO: 97 to the nucleic acid sequence of any one of SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to 229, or SEQ ID NO: 238 to SEQ ID NO: 241, or a variant thereof variant, the variant is 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) contains a 3' flanking region located 3' relative to the leader sequence. In some embodiments, the polynucleotide comprising a stem-loop sequence having SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 46 to SEQ ID NO: 62, SEQ ID NO: 97 to the nucleic acid sequence of any one of SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 138, SEQ ID NO: 226 to 229, or SEQ ID NO: 238 to SEQ ID NO: 241, or a variant thereof variant, the variant is 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) contains a loop region between the leader sequence and the follower sequence, where the loop region contains the microRNA loop sequence.

In some embodiments, the first promoter and/or optionally the 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 including the cytomegalovirus enhancer (e.g., CAG or CBA), U6, H1 and 7SK promoters.

In some embodiments, the 5' flanking region comprises a polynucleotide that shares at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) the nucleic acid sequence of any of the following %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 217, SEQ ID NO: 220 or SEQ ID NO: 223. In some embodiments, the 3' flanking region comprises a polynucleotide that shares at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) the nucleic acid sequence of any of the following %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 218, SEQ ID NO: 221 or SEQ ID NO: 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 that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90% identical to any of the following nucleic acid sequences) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 219, SEQ ID NO: 222 or SEQ ID NO: 225. In some embodiments, the microRNA loop sequence comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 222. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the microRNA loop sequence comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO: 222.

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

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

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

In some embodiments, the performance cartridge further includes one or more (eg, two, three, four, or five) polyadenylation message sequences (eg, to improve the performance cartridges disclosed herein or Nuclear export, translation and stability of inhibitory polynucleotides of vectors). The polyadenylation message sequence can be located 3' and/or 5' of the 3' ITR sequence of the terminal inhibitor polynucleotide sequence (e.g., the ASO sequence disclosed herein, such as a miRNA 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 that 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: SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In some embodiments, the RBG polyadenylation signal includes a polynucleotide having the following nucleic acid sequence: SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 215. In some embodiments, the polyadenylation message is a bovine growth hormone (BGH) polyadenylation message. In some embodiments, the BGH polyadenylation message sequence comprises a polynucleotide that is at least 85% (e.g., at least 86%, 87%, 88%, 89%) identical to the nucleic acid sequence of SEQ ID NO: 216 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the BGH polyadenylation message sequence includes a polynucleotide having the nucleic acid sequence of SEQ ID NO: 216.

In some embodiments, the performance cartridge further contains one or more (eg, two, three, four, or five) filler sequences. In some embodiments, the one or more (e.g., two, three, four, or five) stuffer sequences are located at the 3' end of the expression cassette (e.g., between the polyadenylation sequence and the 3' ITR sequence between). In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences are at least 85% (e.g., at least 86%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 250 , 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four, or five) stuffer sequences are at least 90% (e.g., at least 91%, 95%) identical to the nucleic acid sequence of SEQ ID NO: 250 , 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences are at least 95% (e.g., at least 96%, 97%) identical to the nucleic acid sequence of SEQ ID NO: 250 , 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences have at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, the one or more (e.g., two, three, four, or five) stuffer sequences have the nucleic acid sequence of SEQ ID NO: 250. In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences are at least 85% (e.g., at least 86%, 90%) identical to the nucleic acid sequence of SEQ ID NO: 251 , 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four or five) filler sequences are at least 90% (e.g., at least 91%, 95%) identical to the nucleic acid sequence of SEQ ID NO: 251 , 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences are at least 95% (e.g., at least 96%, 97%) identical to the nucleic acid sequence of SEQ ID NO: 251 , 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the one or more (e.g., two, three, four, or five) filler sequences have at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 251. In some embodiments, the one or more (e.g., two, three, four, or five) stuffer sequences have the nucleic acid sequence of SEQ ID NO: 251.

In some embodiments, a performance cassette of any aspect and embodiment includes, from 5' to 3': (a) a 5' ITR sequence; (b) optionally, an enhancer sequence; (c) ) first promoter sequence; (d) optionally, the intron sequence; (e) polynucleotide containing the stem-loop sequence; (f) optionally, the second promoter sequence; (g) optionally, the polynucleotide containing the stem a polynucleotide of a loop sequence; (h) a polyadenylation message sequence, such as an RBG polyadenylation message sequence; (i) one or more stuffer sequences; and (j) 3' ITR.

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

In some embodiments, the presentation cassette of the aforementioned aspects and embodiments is incorporated into the carrier of the aforementioned 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 system is selected from the group consisting of: adeno-associated virus (AAV), retrovirus, adenovirus, parvovirus, coronavirus, negative-strand RNA virus, orthomyxovirus, rod-shaped virus Viruses, paramyxoviruses, orthostranded RNA viruses, picornaviruses, alphaviruses, double-stranded DNA viruses, herpesviruses, Epstein-Barr virus, cytomegalovirus, fowlpox virus, and canaries Poxvirus. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is an AAV5, AAV9, or AAVrh10 vector.

On the other hand, the present disclosure provides a method for inhibiting the expression of Grik2 in a cell, which method includes contacting the cell with at least one polynucleotide of the aforementioned aspects and embodiments, a vector of the aforementioned aspects and embodiments, or the aforementioned aspects and embodiments. The embodiment shows the cassette contact.

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%.

In some embodiments, the cells are human cells. In some embodiments, the cell is a neuron (eg, human neuron). In some embodiments, the neuron is a hippocampal neuron (eg, human hippocampal neuron). In some embodiments, the hippocampal neuron is a DGC (e.g., human DGC or pyramidal neuron). In some embodiments, the DGCs comprise recurrent mossy fiber axons. The cells may also be neuronal cells derived from induced potentiated stem cells (iPSCs), such as iPSC-derived glutamatergic neurons expressing Grik2 .

In another aspect, the present disclosure provides a method of treating or ameliorating a disease in an individual in need thereof, the method comprising administering to the individual at least one polynucleotide of the aforementioned aspects and embodiments, the aforementioned aspects and embodiments The carrier, or the performance cassette of the aforementioned aspects and embodiments.

In some embodiments, the condition is epilepsy. In some embodiments, the epilepsy is temporal lobe epilepsy (TLE), chronic epilepsy, and/or refractory epilepsy. In some embodiments, the epilepsy is TLE. In some embodiments, the TLE is a lateral TLE (ITLE), such as unilateral TLE and/or bilateral TLE. In some embodiments, the TLE is a mesial TLE (mTLE).

In some embodiments, the subject is a human.

In another aspect, the present disclosure provides a pharmaceutical composition, which includes the polynucleotide of the aforementioned aspects and embodiments, the vector of the aforementioned aspects and embodiments, or the expression cassette of the aforementioned aspects and embodiments, and Pharmaceutically acceptable carriers, diluents or excipients.

In another aspect, the present disclosure provides a kit, which includes the pharmaceutical composition and drug instructions of the aforementioned aspect. In some embodiments, the drug package insert includes instructions for use of the pharmaceutical composition in the aforementioned aspects and methods of the embodiments. definition

For convenience, the following provides the meanings of some terms and phrases used in the description, examples, and appended patent claims. Unless stated otherwise or implied from context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

In this application, unless the context clearly dictates otherwise, (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 "comprising" and "comprising" may be understood to cover the itemized components or steps, whether presented alone or together with one or more additional components or steps.

The term "about" refers to an amount that is ±10% of the quoted value and may be ±5% of the quoted value or ±2% of the quoted value.

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

The terms "5' untranslated region" and "5'UTR" refer to the region of an mRNA molecule (eg, Grik2 mRNA) that is 5' relative to the start codon. This region is important for the regulation of translation initiation. The 5' UTR may not be translated at all, or some of its regions may be translated in some organisms. The transcription start site marks the beginning of the 5' UTR and ends one nucleotide before the start codon. In eukaryotes, the 5' UTR includes the Kozak consensus sequence with the start codon. The 5' UTR can include cis-acting regulatory elements, also known as the upstream open reading frame, which are important in the regulation of translation. This region can also contain upstream AUG codons and stop codons. Given its high GC content, the 5' UTR can form secondary structures such as hairpin loops that play a role in translational regulation.

The term "administering" refers to providing or administering a therapeutic agent (e.g., an inhibitory polynucleotide that binds to Grik2 mRNA and inhibits its expression, or a vector encoding such a polynucleotide, as disclosed herein) to an individual by any effective route. ). Exemplary routes of administration are described herein and below (eg, intracerebroventricular, intrathecal, intraparenchymal, intravenous, and stereotaxic injection).

The term "adeno-associated viral vector" or "AAV vector" refers to vectors derived from adeno-associated virus serotypes, including, but not limited to, 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-TT, AAV-DJ8 or AAV.HSC16. AAV vectors may delete all or part of one or more of the AAV wild-type genes, such as the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences facilitate the rescue, replication, and packaging of AAV virions. Therefore, AAV vectors are defined herein as including at least those cis-sequences required for viral replication and packaging (e.g., a functional ITR). The ITR need not be a wild-type polynucleotide sequence and may be altered, for example, by insertion, deletion, or substitution of nucleotides, so long as the sequences provide functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide at least operably linked components in the direction of transcription, control elements including a transcription initiation region, target DNA (e.g., a polynucleotide encoding an inhibitory RNA agent of the present disclosure) ) and the transcription termination region.

The terms "adeno-associated virus inverted terminal repeat" and "AAV ITR" refer to the art-recognized regions flanking each end of the AAV genome, which together function in cis as DNA replication origins and viral packaging signals. The AAV ITR together with the AAV rep coding region provides for efficient excision and integration into the mammalian genome of the polynucleotide sequence inserted between the two flanking ITRs. The polynucleotide sequence of the AAV ITR region is known. As used herein, "AAV ITR" does not necessarily include wild-type polynucleotide sequences, which may be altered, for example, by insertion, deletion, or substitution of nucleotides. Additionally, AAV ITRs can be derived from any of several AAV serotypes, including, but not limited to, 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-TT, AAV-DJ8 or AAV.HSC16, etc. Furthermore, the 5' and 3' ITRs flanking the selected polynucleotide sequence in the AAV vector need not be the same or derived from the same AAV serotype or isolate, as long as they function as expected, e.g., to allow for the synthesis of host cell genes Excise and rescue the target sequence into the body or vector, and allow the heterologous sequence to be integrated into the recipient cell genome while the AAV Rep gene product is present in the cell. Additionally, AAV ITRs can be derived from any of several AAV serotypes, including, but not limited to, 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-TT, AAV-DJ8 or AAV.HSC16, etc.

The terms "antisense oligonucleotide" and "ASO" refer to inhibitory polynucleotides that are capable of hybridizing to a target mRNA molecule (e.g., Grik2 mRNA) through complementary base pairing and inhibiting through mRNA destabilization and degradation Its performance, or inhibition of translation.

The term "cDNA" refers to a DNA nucleic acid sequence that is identical to an mRNA sequence (ie, uridine is replaced by thymidine). Often, the terms cDNA and mRNA are used interchangeably when referring to a specific gene (eg, the Grik2 gene), as those skilled in the art will understand that the cDNA sequence is the same as the mRNA sequence, except that uridine is read as thymidine. Furthermore, where referring to DNA sequences encoding the antisense constructs disclosed herein or RNA transcripts encoded thereby, the terms "DNA" and "RNA" are used interchangeably to refer to Antisense sequence. Additionally, certain DNA sequences disclosed herein (eg, those encoding Grik2 antisense sequences) may include RNA nucleotides, in which case the entire sequence may be referred to as a "DNA sequence" or "RNA sequence."

The term "coding sequence" corresponds to the nucleic acid sequence of an mRNA molecule encoding a protein or part thereof. Relatedly, "non-coding sequences" correspond to nucleic acid sequences of the mRNA molecule that do not encode proteins or parts thereof. Non-limiting examples of non-coding sequences include 5' and 3' untranslated regions (UTRs), introns, polyadenylate tails, promoters, enhancers, terminators and other cis-regulatory sequences.

The term "complementary" when used to describe a first nucleotide or nucleoside sequence that is related to a second nucleotide or nucleoside sequence refers to a polynucleotide comprising the first nucleotide sequence that under certain conditions is The polynucleotides including the second nucleotide sequence hybridize and form a duplex structure. For example, such conditions may be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C, or 70°C for 12 to 16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual", Sambrook et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may apply, such as physiologically relevant conditions that may be encountered in an organism. Methods to determine the set of conditions most suitable for testing the complementarity of two sequences, depending on the ultimate application of the hybridizing nucleotide or nucleoside, are well known in the art.

As used herein, "complementary" sequences may also include non-Watson-Crick base pairs and/or base pairs formed by or entirely formed from non-natural and alternative nucleotides, so long as the above with respect to their ability to hybridize Requirements are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairing. Complementary sequences between a polynucleotide and a target sequence as described herein include a polynucleotide comprising a first nucleotide sequence and a polynucleotide comprising a second nucleotide sequence in one or both nucleotide sequences. base pairing over its entire length. Such sequences may be referred to herein as "completely complementary" with respect to each other. When a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be completely complementary or they may, upon hybridization to form a duplex of up to 30 base pairs, form a or Multiple, but usually no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to its end application, e.g. Binds to and inhibits the expression of mRNA, such as Grik2 mRNA. For example, a polynucleotide is complementary to at least a portion of a target mRNA if the sequence is substantially complementary to an uninterrupted portion of the target mRNA.

The term "complementary region" refers to a region of an inhibitory polynucleotide that is substantially complementary to all or part of a gene, primary transcript, sequence (e.g., a target sequence), or processed mRNA, so as to interfere with an endogenous gene (e.g., a target sequence) , the performance of Grik2 ). In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches occur in terminal regions, eg, within 5, 4, 3, or 2 nucleotides of the 5'- and/or 3'-terminal end of the inhibitory polynucleotide.

The terms "conservative amino acid substitution", "conservative substitution" and "conservative mutation" refer to the substitution of one or more amino acids for one or more different amino acids that exhibit similar physical chemistry Properties such as polarity, electrostatic charge, and three-dimensional volume. These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below. Table 1. Representative physicochemical properties of naturally occurring amino acids amino acids 3 letter code 1 letter code Side chain polarity Electrostatic properties at physiological pH (7.4) Three-dimensional volume ^† alanine Ala A non-polar neutral Small Arginine Arg R polarity Cationicity big aspartic acid Asn N polarity neutral medium aspartic acid Asp D polarity Anionic medium cysteine Cys C non-polar neutral medium glutamate Glu E polarity Anionic medium Glutamine gnc Q polarity neutral medium glycine Gly G non-polar neutral Small Histidine His H polarity Both neutral and cationic forms are in equilibrium at pH 7.4 big isoleucine Ile I non-polar neutral big Leucine Leu L non-polar neutral big lysine Lys K polarity Cationicity big methionine Met M non-polar neutral big Phenylalanine Phe F non-polar neutral big proline Pro P non-polar neutral medium Serine Ser S polarity neutral Small threonine Thr T polarity neutral medium Tryptophan tp W non-polar neutral huge tyrosine Tyr Y polarity neutral big Valine Val V non-polar neutral medium ^† Based on volume in A ³ : 50 to 100 is small, 100 to 150 is medium, 150 to 200 is large, and >200 is huge

It can be seen from the table that the retained amino acid family includes (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; (vi) F, Y and W. Thus, a retention mutation or substitution is the substitution of an amino acid for a member of the same amino acid family (for example, Ser for Thr or Lys for Arg).

The phrase "contacting a cell with an inhibitory polynucleotide", such as the inhibitory polynucleotides disclosed herein, includes contacting the cell by any possible means. Contacting the cell with the inhibitory polynucleotide includes contacting the cell with the inhibitory polynucleotide in vitro or contacting the cell with the inhibitory polynucleotide in vivo. Contacting a cell with an inhibitory polynucleotide may also refer to contacting a cell with a nucleic acid vector encoding the inhibitory polynucleotide or a pharmaceutical composition comprising the nucleic acid vector. Contact may be direct or indirect. Thus, for example, an inhibitory polynucleotide can be placed in physical contact with a cell by an individual performing the method, or alternatively, an inhibitory polynucleotide agent can be placed in a situation that permits or results in subsequent contact with the cell. Contacting cells in vitro can be accomplished by incubating the cells with an inhibitory polynucleotide. Contacting cells in vivo can be accomplished, for example, by injecting an inhibitory polynucleotide into or near the tissue where the cell is located, or by injecting an inhibitory polynucleotide agent into another area, such as the bloodstream or subcutaneous space, such that the agent It then reaches the tissue where the cells to be contacted are located. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be contacted with inhibitory polynucleotides ex vivo and subsequently transplanted into an individual.

Contacting the cell with the inhibitory polynucleotide includes "introducing or delivering the inhibitory polynucleotide into the cell" by promoting or affecting uptake or uptake into the cell. Uptake or uptake of an inhibitory polynucleotide or a nucleic acid vector encoding the inhibitory polynucleotide can occur by unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the inhibitory polynucleotide into the cell can be in vitro and/or in vivo. For example, for in vivo introduction, the inhibitory polynucleotide can be injected into the tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. In another example, an inhibitory polynucleotide can be introduced into a cell by transduction, such as by a viral vector encoding the inhibitory polynucleotide. Viral vectors can undergo cellular processing (e.g., cellular internalization, capsid shedding, transcription of the inhibitory polynucleotide, and processing by Drosha and Dicer) to express the encoded inhibitory polynucleotide. Further methods are described below and/or are known in the art.

The terms "disrupt expression,""inhibitexpression," or "reduce expression" with respect to a gene (eg, Grik2 ) refer to preventing or reducing the formation of a functional gene product (eg, GluK2 protein). A gene product is functional if it fulfills 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 interfering RNA molecules (eg, ASOs), such as those described herein.

The terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein mean sufficient to affect a beneficial or The number of desired outcomes (including clinical outcomes). Therefore, "effective amount" or its synonyms depend on the context in which it is applied. For example, in the case of treating temporal lobe epilepsy (TLE), an effective amount of a composition, vector construct, or virus is sufficient to achieve a therapeutic response as compared to the response obtained without administration of the composition, vector construct, or viral vector Amount of carrier. The amount corresponding to a given composition described herein will vary depending on a variety of factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder and its severity, the identity of the individual (e.g., age, gender, weight), the host being treated, and/or in the case of epilepsy, the size of the epileptic focus (eg, brain volume), etc., but can still be determined according to methods commonly known in the art. Additionally, 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 an individual 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 optimal therapeutic response.

The term "epilepsy" refers to one or more neurological disorders characterized by recurrent epileptic seizures. Epilepsy can be classified based on electroclinical symptoms according to the classification and terminology of the International League Against Epilepsy (ILAE; Berg et al., 2010 ). These symptoms can be classified according to age of onset, a unique group of epilepsy (operative syndromes) and structural metabolic causes, such as: (A) Age of onset: (i) Neonatal period, including benign familial neonatal epilepsy (BFNE), Early myoclonic encephalopathy (EME), Ohtahara syndrome; (ii) infancy, including infantile epilepsy with metamorphic focal seizures, West syndrome, myoclonic epilepsy of infancy (MEI), benign infantile epilepsy, Benign familial infantile epilepsy, Dravet syndrome, myoclonic encephalopathy in non-progressive disease; (iii) childhood, including febrile seizure plus (FS+), Panayiotopoulos syndrome, epilepsy with myoclonic atonia (formerly Seizures, benign epilepsy with centrotemporal spikes (BECTS), autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), late-onset childhood occipital epilepsy (Gastaut type), epilepsy with myoceles Absence syndrome, Lennox-Gastaut syndrome, epileptic encephalopathy with continuous spikes during sleep (CSWS), Landau-Kleffner syndrome (LKS), childhood absence epilepsy (CAE); (iv) adolescence to adulthood, including adolescent absence epilepsy Epilepsy (JAE), juvenile myoclonic epilepsy (JME), epilepsy with generalized tonic epilepsy only, progressive myoclonic epilepsy (PME), autosomal dominant epilepsy with auditory features ( ADEAF), other familial temporal lobe epilepsies; (v) different ages of onset, including familial focal epilepsy with variable foci (children to adults), reflex epilepsy; (B) a unique group of epilepsy (operative syndrome ) Including mesial temporal lobe epilepsy (MTLE), Rasmussen syndrome, gigolo-like seizures with hypothalamic hamartomas, hemiconvulsions-hemiplegia-epilepsy; (C) Epilepsy caused and organized by structural metabolic causes including malformations of cortical development ( Hemimegalencephaly, ectopia, etc.), neurocutaneous syndromes (tuberous sclerosis complex and Sturge-Weber syndrome), tumors, infections, trauma, hemangioma, perinatal injuries, and stroke. The term “refractory epilepsy” refers to epilepsy that is medically refractory; that is, current medical treatments are ineffective in treating the patient's disease (see, for example, Dario J. Englot et al., 2013).

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

When used in the context of the expression of genes or nucleic acids, the term "expression" refers to the conversion of information contained in a gene into a gene product. A gene product can be a direct transcription product of a gene (such as mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of the mRNA. Gene products also include mRNA modified by processes such as capping, polyadenylation, methylation, and editing, and by processes such as methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP - Ribosylated, myristoylated and glycosylated proteins (e.g. GluK2).

The term "expression" refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping formation and/or 3' end processing); (3) translation of RNA into polypeptides or proteins; and (4) post-translational modification of polypeptides or proteins. Expression of a target gene in an individual can be demonstrated, 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 individual (e.g., using methods described herein or known in the art). As assessed by known RNA detection procedures, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), the corresponding decrease or increase in the amount or concentration of the protein (e.g., using proteins described herein or known in the art detection method, such as enzyme-linked immunosorbent assay (ELISA), etc.), and/or a decrease or increase in the activity of the corresponding protein (e.g., in the case of ion channels, as described herein or as described in the art. assessed by known electrophysiological methods).

The term "GluK2", also known as "GluR6", "GRIK2", "MRT6", "EAA4" or "GluK6", refers to the glutamate ionotropic receptor kainate type subunit 2 protein, as in Named according to the currently used IUPHAR nomenclature (Collingridge, GL, Olsen, RW, Peters, J., Spedding, M., 2009. A nomenclature for ligand-gated ion channels. Neuropharmacology 56, 2-5). The terms "GluK2-containing KAR,""GluK2receptor,""GluK2protein" and "GluK2 subunit" are used interchangeably throughout the text and generally refer to the protein encoded or expressed by the Grik2 gene.

The terms "guide strand" and "guide sequence" refer to components of a stem-loop RNA structure (e.g., shRNA or microRNA) located on the 5' or 3' stem-loop arm of the stem-loop structure, where the guide strand/sequence includes Grik2 mRNA antisense sequence (e.g., any of the following: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245, or a nucleic acid sequence having at least Variants with 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245), this antisense sequence can bind to Grik2 mRNA and inhibit its expression. The leader/sequence may also include additional sequences, such as spacer sequences or linker sequences. The guide sequence may be complementary or substantially complementary (eg, with no more than 7, 6, 5, 4, 3, 2, or 1 mismatch) to the following strands/sequences of the stem-loop RNA structure.

The term "ionotropin glutamate receptor" includes NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isothiazopropionic acid) ) and members of the kainate receptor (KAR) class. Functional KARs can be assembled into tetrameric assemblies from homologous or heterologous combinations of five subunits named GluK1, GluK2, GluK3, GluK4, and GluK5 subunits (Reiner et al., 2012). In some cases, the target of the present disclosure is the KAR complex consisting of GluK2 and GluK5. Inhibition of the Grik2 gene is sufficient to eliminate GluK2/GluK5 kainate receptor function, as it was observed that the GluK5 subunit itself does not form a functional homologous channel.

"Performance inhibitor" refers to an agent (eg, inhibitory RNA agent of the present disclosure) that has a biological effect to inhibit or reduce the expression of a gene (eg, Grik2 gene). The expression of a suppressor gene (eg, Grik2 gene) usually results in the reduction or even elimination of the gene product (protein, eg, GluK2 protein) in the target cell or tissue, but varying levels of inhibition may be achieved. Inhibiting or reducing performance is often called knockdown.

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 speaking, an "isolated" polynucleotide refers to a polynucleotide that is artificial, chemically synthesized, purified, and/or heterologous to the nucleic acid sequence from which the polynucleotide was obtained.

The term “microRNA” refers to short (e.g., typically 22 nucleotides) non-coding RNA sequences that regulate the translation of mRNA and thus affect the abundance of target proteins. Some microRNAs are transcribed from single, monocistronic genes, whereas others are transcribed as part of polycistronic gene clusters. The structure of microRNA can include 5' and 3' flanking sequences, hairpin sequences including stem and loop sequences. During intracellular processing, immature microRNAs are truncated by Drosha, which excises 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 at the level of translational regulation by binding to regions of the mRNA molecule (usually the 3' untranslated region) and causing cleavage, degradation, instability and/or inefficient translation of the mRNA. Binding of a microRNA to its target is typically mediated by a short (e.g., 6 to 8 nucleotides) "seed region/sequence" within the microRNA hairpin sequence. Throughout this disclosure, the term siRNA may include its miRNA equivalents, such that a miRNA encompasses the same bases with homology to the target (e.g., in the seed region) as its 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.

The term "nucleotide" is defined as a modified or naturally occurring deoxyribonucleotide or ribonucleotide. Nucleotides generally include purines and pyrimidines, including thymidine, cytidine, guanosine, adenosine, and uridine. As used herein, the term "inhibitory polynucleotide" is defined as an oligomer of a nucleotide as defined above or a modified nucleotide disclosed herein. The term "inhibitory polynucleotide" refers to a 3'-5' or 5'-3' oriented nucleic acid sequence, which may be single-stranded or double-stranded. Inhibitory polynucleotides used in the context of the present disclosure may specifically be DNA or RNA. The term may also include "inhibitory polynucleotide analogs," which refers to inhibitory polynucleotides that have, for example, (i) a modified backbone structure, e.g., that is different from that found in native oligonucleotides and The backbone of the standard phosphodiester bonds found in polynucleotides, and (ii) optionally having modified sugar moieties, for example, an N-morpholinyl moiety instead of a ribose or deoxyribose moiety. Inhibitory polynucleotide analogs support bases capable of hydrogen bonding with standard polynucleotide bases via Watson-Crick base pairing, where the analog backbone allows the inhibitory polynucleotide analog molecule to bond with the standard The bases of a polynucleotide (eg, single-stranded RNA or single-stranded DNA) exhibit hydrogen bonds between the bases in a sequence-specific manner. Specifically, analogs are those having a substantially uncharged phosphorus-containing backbone. The substantially uncharged phosphorus-containing backbone of the inhibitory polynucleotide analog is a backbone in which a majority of the subunits are bonded, e.g., between 50% and 100%, typically at least 60% to 100% or 75% Or 80% of its linkages are uncharged and contain a single phosphorus atom. Furthermore, the term "inhibitory polynucleotide" may include inhibitory polynucleotide sequences that are inverted relative to their normal direction of transcription and thus correspond to an RNA or DNA sequence that is complementary to a target gene mRNA molecule expressed within a host cell . The antisense leader can be constructed in a number of different ways, provided it interferes with the expression of the target gene. For example, an antisense leader can be constructed by being reverse complementary to the coding region (or a portion thereof) of a target gene in the opposite direction relative to its normal direction of transcription, allowing for the transcription of its complement, (e.g., from the antisense gene and The RNA encoded by the sense gene may be complementary). The inhibitory polynucleotide does not need to have the same intron or exon pattern as the target gene, and non-coding segments of the target gene can be as effective as coding segments such as ASO in achieving antisense inhibition of target gene expression. Equally valid. In some cases, the inhibitory RNA has the same exon pattern as the target gene.

The inhibitory polynucleotide can be of any length that allows targeting and hybridization to Grik2 mRNA (e.g., the inhibitory polynucleotide is perfectly or substantially complementary to at least one region of Grik2 mRNA) and can be about 10 to 50 bases Base pair ranges in length, for example, about 15 to 50 base pairs in length or about 18 to 50 base pairs in length, for example, 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, such as about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24 , 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24 ,19 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 in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

The terms "traveler strand" and "traveler sequence" refer to components of a stem-loop RNA structure (e.g., shRNA or microRNA) located on the 5' or 3' stem-loop arm of the stem-loop structure, including those that are antisense to Grik2 mRNA Sequences that are complementary or substantially complementary (e.g., have no more than 7, 6, 5, 4, 3, 2, or 1 mismatch) (e.g., any of the following: SEQ ID NO: 16 to SEQ ID NO : 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 , and SEQ ID NO: 242 to SEQ ID NO: 245, or it shares at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%) with the nucleic acid sequence of any of the following %, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245). The companion strand/sequence may also include additional sequences, such as spacer sequences or linker sequences. The follower sequence may be complementary or substantially complementary to the leader/sequence of the stem-loop RNA structure.

The term "plastid" 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 has been linked. Certain plastids are able to replicate autonomously in the host cell into which they are introduced (e.g., bacterial plastids with bacterial origins of replication, and episomal mammalian plastids). Other vectors (e.g., non-episomal mammalian vectors) can be introduced into a host cell and integrated into the host cell's genome, thereby replicating with the host genome. Certain plastids are capable of directing the expression of genes to which they are operably linked. As used herein, "gene" refers to a polynucleotide encoding a protein, microRNA, siRNA, shRNA, shmiRNA, and further includes one or more regulatory sequences (e.g., promoter, enhancer, intron, termination sequence, etc. ).

The term "promoter" refers to the recognition site on DNA that is bound by RNA polymerase. Polymerase drives the transcription of polynucleotides. Exemplary promoters suitable for use with 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 is a nucleic acid regulatory element. Additionally, the term "promoter" may refer to synthetic promoters, which are regulatory DNA sequences that do not occur naturally in biological systems. Synthetic promoters contain naturally occurring promoter portions combined with polynucleotide sequences that do not occur in nature, and can be optimized for expression of recombinant DNA using a variety of polynucleotides, vectors, and target cell types.

"Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the number of nucleotides in the candidate sequence that are identical to the reference polynucleotide after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percent sequence identity. The percentage of nucleic acids or amino acids in an acid or polypeptide sequence that are identical. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be accomplished by a variety of means within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2 or Megalign software. Appropriate parameters for aligning sequences can be determined using generally accepted and customary methods, including any algorithms required to achieve maximal 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. As an example, the percentage of sequence identity of a given nucleic acid or amino acid sequence A with or against a given nucleic acid or amino acid sequence B (can also be expressed as a certain percentage of sequence identity with or against a given nucleic acid or amino acid sequence B Sequence identity for a given nucleic acid or amino acid sequence A) is calculated as follows: 100 times (score X/Y) where The number of identical matching nucleotides or amino acids, and where Y is the total number of nucleic acids in B . When the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity between A and B will not be equal to the percent sequence identity between B and A. Regardless of the percent sequence identity between the candidate sequence and the reference polynucleotide or polypeptide sequence, the candidate sequence retains at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85 A reference polynucleotide that is %, 90%, 95%, 97%, 99% or 100% functional (e.g., the ability to reduce the level of Grik2 mRNA as defined herein or the expression level of GluK2 protein as defined herein) or polypeptide sequence.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are suitable for contact with tissue of an individual such as a mammal (e.g. human, human) without undue toxicity, irritation, allergic reaction and other problematic complications, commensurate with a reasonable benefit/risk ratio.

As used herein, the term "pharmaceutical composition" means a composition containing a compound described herein (e.g., an inhibitory nucleic acid molecule (e.g., RNA) or a vehicle containing the compound) formulated together with a pharmaceutically acceptable excipient, and may, in some cases, be manufactured or sold with the approval of government regulatory agencies as part of a treatment regimen to treat a disease in mammals. Pharmaceutical compositions may be formulated, for example, in unit dosage form for oral administration (e.g., tablets, capsules, caplets, gel capsules, or syrups), for topical administration (e.g., as creams, gels, lotions, agent or ointment), intravenous administration (e.g., as a sterile solution without particulate plugs and in a solvent system suitable for intravenous use), intrathecal injection, intracerebroventricular injection, intraparenchymal injection, or any other pharmaceutically acceptable Acceptance of concoctions.

"Pharmaceutically acceptable excipient" refers to any ingredient other than a compound described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is substantially non-toxic and non-inflammatory to the patient. Excipients may include, for example: anti-adhesive agents, antioxidants, adhesives, coating agents, compression aids, disintegrating agents, dyes (pigments), emollients, emulsifiers, fillers (diluents), Film-forming agents or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose, crospolyvinylpyrrolidone, citric acid, crosslinked Dipovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, formazan Cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, polyvinylpyrrolidone, pregelatinized starch, propylparaben, retinyl palmitate, insecticides Gum, 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.

The compounds described herein (e.g., inhibitory nucleic acid molecules (e.g., RNA) and vectors containing the same) may have ionizable groups to enable preparation of pharmaceutically acceptable salts. Such salts may be acid addition salts involving inorganic or organic acids, or in the case of acidic forms of the compounds described herein, such salts may be prepared from inorganic or organic bases. Typically, the compounds are prepared or used as pharmaceutically acceptable salts, or as pharmaceutically acceptable acid or base addition products. Suitable pharmaceutically acceptable acids and bases, as well as methods for preparing suitable salts, are well known in the art. Salts can be prepared from pharmaceutically acceptable to nontoxic 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, cyclopentane propionate, digluconate, lauryl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate , hemisulfate, enanthate, caproate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate , malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, bis Hydroxylate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, Tartrate, thiocyanate, tosylate, undecanoate and valerate. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium and magnesium, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine , dimethylamine, trimethylamine, triethylamine and ethylamine.

The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (such as polyadenylation message sequences) that control the transcription or transfer of a gene. Such regulatory sequences are described, for example, in Perdew et al., Regulation of Gene Expression (Humana Press, New York, NY, (2014)); incorporated herein by reference.

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

The terms "short interfering RNA" and "siRNA" refer to inhibitory polynucleotides containing double-stranded nucleic acids, where each strand contains RNA, RNA analogs, or RNA and DNA. siRNA molecules can include between 19 and 23 nucleotides (eg, 21 nucleotides). siRNA typically has a 2 bp overhang at the 3' end of each strand, such that the duplex region in the siRNA contains 17 to 21 nucleotides (eg, 19 nucleotides). Typically, the antisense strand of siRNA is fully complementary to the target sequence of the target gene/RNA. siRNA molecules operate within the RNA interference pathway, inhibiting mRNA expression by binding to target mRNA (eg, Grik2 mRNA) and degrading it through Dicer-mediated mRNA cleavage. Throughout this disclosure, the term siRNA is intended to include its equivalents, such that a miRNA encompasses the same base homology to the target as its equivalent siRNA.

The terms "short hairpin RNA" and "shRNA" refer to inhibitory polynucleotides containing single-stranded RNAs of 50 to 100 nucleotides that form a stem-loop structure in cells containing 5 to 30 nucleotides. A loop region of nucleotides, and 15 to 50 nucleotides long complementary RNAs on both sides of the loop region, which form a double-stranded stem through base pairing between the complementary RNA sequences; and, in some cases, an additional 1 to 500 nucleotides included before and after each complementary strand forming the stem. For example, shRNA often requires specific sequences 3' of the hairpin to terminate transcription by RNA polymerase. These shRNAs often bypass Drosha processing because they contain short 5' and 3' flanking sequences. Other shRNAs, such as "shRNA-like microRNAs" transcribed from RNA polymerase II, include longer 5' and 3' flanking sequences and require processing by Drosha in the nucleus, followed by export of the cleaved shRNA from the nucleus to the cytoplasm liquid and further cleaved by Dicer in the cytosol. Like siRNA, shRNA binds to the target mRNA in a sequence-specific manner, thereby cleaving and destroying the target mRNA and thereby inhibiting the expression of the target mRNA.

As used herein, the terms "specifically hybridize" and "specifically bind" refer to a sufficient degree of complementarity between a polynucleotide and a target nucleic acid (e.g., Grik2 mRNA) to induce a desired effect (e.g., decrease or inhibit GluK2 expression from Grik2 mRNA), while showing minimal or no effect on non-target nucleic acids. Specific hybridization or binding can occur under physiological conditions. For example, when the number of nucleobases in a polynucleotide (e.g., an antisense polynucleotide) that is complementary to a nucleobase corresponding to a target nucleic acid (e.g., an mRNA sequence) facilitates the fitting of the polynucleotide to the The target nucleic acid does not fit to the non-target nucleic acid (e.g., complementarity corresponds to, e.g., the percent sequence identity of the binding portion of the polynucleotide to the target nucleic acid is 80% or greater (e.g., 85%, 90 %, 95%, 97%, 99% or 100%)), specific hybridization or binding occurs. Those skilled in the art will understand that in this case, the nucleic acid sequence in the polynucleotide (e.g., antisense oligomer) has a high degree of complementarity (e.g., at least about 80%, 85%) to the nucleic acid sequence in the target nucleic acid. %, 90%, 95%, 97%, 99%, or 100% complementary, such as over a defined number of polynucleotides (eg, about 7 to 22 nucleobases)).

The terms "individual" and "patient" refer to animals (eg, mammals, such as humans). An individual to be treated according to the methods described herein may be an individual who has been diagnosed with epilepsy (e.g., TLE), or is at risk of developing the condition. Diagnosis can be performed by any method or technique known in the art. Individuals to be treated in accordance with the present disclosure may have undergone standard testing or may have been identified without testing as being at risk due to the presence of one or more risk factors associated with the disease or condition.

The term "temporal lobe epilepsy" or "TLE" refers to a chronic neurological disorder characterized by chronic and recurrent epileptic seizures (epilepsy) originating in the temporal lobe of the brain. This disease differs from acute epileptic seizures in native brain tissue because TLE is characterized by morphological and functional reorganization of neuronal networks and recurrent mossy fiber sprouting from granule cells in the dentate gyrus of the hippocampus, whereas acute epileptic seizures in native tissue Seizures do not trigger this circuit-specific reorganization. TLE may be due to the development of epileptic lesions in one or both hemispheres of the brain.

The terms "transduction" and "transduction" refer to the introduction of a nucleic acid material (e.g., a vector, such as a viral vector construct or a portion thereof) into a cell and the subsequent expression within the cell of the nucleic acid material (e.g., a vector construct or a portion thereof). Part) Methods for encoding polynucleotides.

The term "treatment" or "treatment" refers to preventive and preventive treatment as well as curative or disease-modifying treatment, including the treatment of patients who are at risk of or suspected of having contracted a disease, and the treatment of patients who are ill or have been diagnosed with a disease or medical condition of patients. Treatment also includes suppression of clinical relapse. Treatment may be administered to an individual who has a medical condition or is at risk of eventually acquiring the condition in order to prevent, cure, delay the onset of the condition, reduce the severity of the condition, or ameliorate one or more symptoms of the condition or recurrence of the condition, or to prolong the individual's life Survival beyond that expected in the absence of such treatment. "Treatment regimen" refers to the mode of treatment for a disease, such as the mode of administration used during treatment. Treatment options may include induction and maintenance options. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The overall goal of an induction regimen is to provide patients with a high level of medication during the initial stages of the treatment regimen. Induction regimens may (partly or fully) use a "loading regimen," which may include administering higher doses of drugs than what your doctor uses during the maintenance regimen, and giving drugs more frequently than your doctor will give during maintenance therapy. , or both. The phrase "maintenance regimen" or "maintenance phase" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during treatment of a disease, e.g., to keep the patient in remission for an extended period of time (months or years). Maintenance regimens may employ continuous therapy (e.g., administration of medication at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinuation of therapy, intermittent therapy, treatment upon relapse or upon achievement of certain predetermined criteria (e.g., , disease manifestation) post-treatment).

The term "vector" includes nucleic acid vectors, for example DNA vectors such as plasmids, RNA vectors or another suitable replicon (eg a viral vector). A variety of vectors have been developed for delivering polynucleotides encoding exogenous polynucleotides or proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994/011026; this patent is incorporated herein by reference as it relates to vectors suitable for expressing target nucleic acid material. Expression vectors suitable for use in the compositions and methods described herein contain polynucleotide sequences as well as additional sequence elements, for example, for expression of heterologous nucleic acid materials (e.g., ASOs) in mammalian cells. Certain vectors useful for expressing 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 expressing inhibitory nucleic acid (e.g., RNA) agents disclosed herein contain polynucleotide sequences that increase the rate of translation of such polynucleotides or increase the nucleic acid produced by gene transcription. (e.g., RNA) stability or nuclear export. Such sequence elements include, for example, 5' and 3' untranslated regions, IRES, and polyadenylation message sequence sites in order to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, commycin, nourseothricin or geomycin.

As used herein, the term "variant" refers to a sequence modified by a starting sequence (e.g., a reference sequence) that reasonably includes one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides. The inhibitory polynucleotide sequence of the present disclosure or its complement (eg, its substantial or complete complement) obtained by acid modification (substitution, insertion, deletion or mismatch). Such modifications may improve at least one characteristic (e.g., biological function) of the polynucleotide (e.g., enhance RISC loading or retention of the leader strand, decrease RISC loading or retention of the follower strand, or increase the ratio of leader to strand production, and Enhance inhibition of target nucleic acid sequences).

sequence list

This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on April 28, 2022, named "51460-007WO3_Sequence_Listing_4_28_22_ST25", and has a size of 297,127 bytes.

Described herein is the use of inhibitory polynucleotides with modifications (e.g., polynucleotides encoding inhibitory RNA agents) to treat epilepsy, such as temporal lobe epilepsy (TLE; e.g., treatment refractory) in an individual (such as a mammalian individual, e.g., a human) (e.g., enhance) RNA-induced silencing complex (RISC) loading, and, e.g., enhance the production of antisense leader strands and minimize the production of follower strands, This promotes greater knockdown of Grik2 mRNA and GluK2 expression and reduces the potential risk of follower-induced off-target effects and toxicity. For example, a therapeutically effective amount of an inhibitory RNA molecule targeting the mRNA encoded by the glutamate ionotropic receptor kainate type subunit 2 ( Grik2 ) gene (e.g., Antisense oligonucleotides (ASOs), shRNA, siRNA, shmiRNA, or nucleic acid vectors encoding them, such as those described herein), to treat epilepsy in individuals (eg, humans) in need thereof. Also described herein are compositions containing nucleic acid vectors (eg, viral vectors, such as adeno-associated virus (AAV) vectors) encoding inhibitory RNA agents that target Grik2 mRNA for use in the treatment of TLE. Grik2

Grik2 is a 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 kainic acid. GluK2-containing kainate receptors (KAR), like other ionotropic glutamate receptors, exhibit rapid ligand gating by glutamate by opening cation channel pores permeable to sodium and potassium to play a role. The KAR complex can be assembled from multiple subunits into heteropolymer or homopolymer assemblies of KAR subunits. This class of receptors is characterized by an extracellular N-terminus and a large peptide loop that together form the ligand-binding domain, and an intracellular C-terminus. The ionotropic glutamate receptor complex itself acts as a ligand-gated ion channel and upon binding glutamate mediates the passage of charged ions across neuronal membranes. Typically, KARs are multimeric assemblies of GluK1, GluK2, and/or GluK3 (previously named 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 the KAR complex are typically determined by RNA splicing and/or RNA editing of the mRNA encoding a specific KAR subunit (eg, adenosine deaminase converts adenosine to inosine). Furthermore, such RNA modifications may affect receptor properties, for example, altering the calcium permeability of the channel. Increased kainate receptor activity is known to be epileptogenic. GluK2-containing KARs are suitable targets to modulate ionotropic glutamate receptor activity and subsequently improve symptoms associated with epileptogenesis (Peret et al., 2014). temporal lobe epilepsy

Epileptogenesis is the process that leads to the establishment of epilepsy, and it may be latent when cellular, molecular, and morphological changes lead to the occurrence of pathological neuronal network reorganization. TLE has been characterized into two main types based on the anatomical origin of the epileptogenic zone. TLEs that originate from the medial temporal lobe (e.g., hippocampal gyrus, parahippocampal gyrus, inferior hippocampal peduncle, amygdala, etc.) are named medial TLE (mTLE), while TLEs that originate from the lateral temporal lobe (e.g., temporal neocortex) are named Lateral TLE (lTLE). Other features of TLE may include neuronal cell death in the CA1, CA3, dentate hilus, and dentate gyrus (DG) regions of the hippocampus; reversal of GABA reversal potential; granule cell (GC) dispersion in the DG; and recurrent Sprouting of GC mossy fibers that lead to the formation of pathophysiological recurrent excitatory synapses (rMF-DGC synapses) on dentate GCs.

Multiple causative factors contribute to the etiology of TLE, including mesial temporal lobe sclerosis, traumatic brain injury, brain infections (eg, encephalitis and meningitis), anoxic brain injury, stroke, brain tumors, genetic syndromes, and Febrile convulsions. Because CNS plasticity depends on both developmental status and brain region-specific susceptibility, not all individuals with brain damage develop epilepsy. The hippocampus, including the DG, has been identified as a brain region particularly susceptible to damage leading to TLE and, in some cases, has been implicated in treatment-resistant (i.e., refractory) epilepsy (Jarero-Basulto, J.J., et al. Pharmaceuticals , 2018, 11, 17; doi:10.3390/ph11010017). Amplification of excitatory glutamatergic signaling may contribute to spontaneous epileptic seizures (Kuruba, et al. Epilepsy Behav. 2009, 14 (Suppl. 1), 65-73).

Without wishing to be bound by theory, abnormal rMF-DGC synapses acting via KARs containing ectopic GluK2 (Epsztein et al., 2005; Artinian et al., 2011, 2015) may play a key role in chronic seizures in TLE ( Peret et al., 2014). For example, in transgenic mice lacking the GluK2 receptor subunit or in the presence of pharmacological agents that inhibit GluK2/GluK5 receptors, interictal spikes and ictal events (i.e., electrophysiology of epileptiform brain activity characteristics) (Peret et al., 2014; Crépel and Mulle, 2015). Although it is feasible to knock down or silence GluK2 in transgenic animal models designed to test these theories, designing an inhibitor that is 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 pattern of gene expression in the brain for homologous and heterologous ionotropic and metabotropic glutamate receptors further complicates any therapeutic strategy. The methods and compositions disclosed herein are suitable for treating TLE (e.g., mTLE or lLTE) by targeting Grik2 mRNA and reducing (e.g., knocking down) the expression of GluK2-containing KARs in neurons or stellate cells. Promotes, for example, the reduction of spontaneous epileptiform discharges in neuronal circuits (eg, hippocampal circuits). Accordingly, the compositions and methods described herein target the physiological causes of disease and can be used in therapy. Inhibitory polynucleotides targeting Grik2 mRNA

Clinical management of TLE is difficult, with at least one-third of TLE patients unable to adequately control debilitating seizures with existing medications. Such patients often experience recurrent epileptic seizures that are difficult to treat. In such cases, TLE patients may undergo invasive and irreversible surgery to remove epileptogenic lesions in the temporal lobes, which may lead to unnecessary cognitive deficits. Therefore, a large proportion of TLE patients require new therapeutic avenues to treat drug-resistant TLE. The compositions and methods described herein provide the benefit of treating the underlying molecular pathophysiology that contributes to the development and progression of TLE.

The compositions described herein are polynucleotides encoding inhibitory nucleic acid constructs (e.g., inhibitory RNA agents or nucleic acid vectors encoding the same) that target Grik2 mRNA (e.g., any of : SEQ ID NO: 164 to SEQ ID NO: 174), such compositions may 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 with any type of TLE (eg, TLE with focal seizures, TLE with generalized seizures, mTLE, or lTLE). Additionally, the presently disclosed methods and compositions may be used to treat TLE due to any etiology, such as mesial temporal lobe sclerosis, traumatic brain injury, brain infections (e.g., encephalitis and meningitis), anoxic brain injury, Stroke, brain tumors, genetic syndromes, or febrile seizures. The compositions and methods described herein may also be administered as preventive treatment to individuals at risk of developing TLE, eg, individuals in the latent phase of TLE progression.

According to the methods and compositions disclosed herein, inhibitory nucleic acids (e.g., inhibitory RNA agents) can be produced by causing cells (e.g., neurons, such as hippocampal neurons, such as hippocampal neurons of the dentate gyrus, such as dentate granules) to Degradation of Grik2 mRNA in glutamatergic cells (DGCs) or glutamatergic pyramidal neurons) inhibits the expression of GluK2, thereby preventing the translation of the mRNA into functional GluK2 protein.

The inhibitory nucleic acid molecules (eg, inhibitory RNA agents) targeting Grik2 mRNA disclosed herein can be used to reduce the frequency of epileptic brain activity (eg, epileptiform discharges) in one or more brain regions or to completely suppress epilepsy. The occurrence of sexual brain activity. Such brain regions may include, but are not limited to, the medial temporal lobe, lateral temporal lobe, frontal lobe, or more specifically, the hippocampus (eg, DG, CA1, CA2, CA3, inferior peduncle) or neocortex. Due to the abnormal expression of GluK2-containing KARs in rMF-DGCs in the DG, the occurrence of epileptic brain activity in the DG may be inhibited.

Accordingly, the present disclosure provides methods for reducing epilepsy 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) or a nucleic acid vector encoding the inhibitory nucleic acid molecule. Methods and compositions for discharge, the inhibitory nucleic acid molecule having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 except SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229, and SEQ ID NO: 238 to SEQ ID NO: 241, the nucleic acid vector, such as SEQ ID NO: 256, has at least 85% (e.g., at least 85 %, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', a miR-30 guide sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%) identical to SEQ ID NO: 19 , 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence that has at least 85% sequence identity with SEQ ID NO: 4; and a miR-30 follower A sequence that is at least 85% identical to SEQ ID NO: 34. In some embodiments, the nucleic acid molecule includes: from 5' to 3', a miR-30 leader sequence having nucleic acid sequence identity with SEQ ID NO: 19; a miR-30 stem-loop sequence having SEQ ID NO: 4 The nucleic acid sequence of SEQ ID NO: 34; and the miR-30 follower sequence, which has the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the nucleic acid molecule includes: a nucleic acid sequence having at least 85% sequence identity from 5' to 3' to SEQ ID NO: 4. In some embodiments, the nucleic acid molecule includes: from 5' to 3', the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid molecule includes: from 5' to 3', a miR-218-1 guide sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that is at least 85% (e.g., at least 85%) identical to SEQ ID NO: 135 %, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence having SEQ ID NO: 147 At least 85% sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', a miR-218-1 guide sequence, which has the nucleic acid sequence of SEQ ID NO: 141; a miR-218-1 stem loop sequence, which has the SEQ ID NO : the nucleic acid sequence of SEQ ID NO: 135; and the miR-218-1 follower sequence, which has the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the nucleic acid molecule includes: from 5' to 3', a nucleic acid sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) identical to SEQ ID NO: 135 , 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', SEQ ID NO: 135.

In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a miR-30 sequence guide sequence that is at least 85% (e.g., at least 85%, 90%, 95%) identical to SEQ ID NO: 19 %, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence that has at least 85% sequence identity with SEQ ID NO: 4; and A miR-30 companion sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%) identical to SEQ ID NO: 34 ) identity; and (b) a miR-218-1 guide sequence that is at least 85% identical to SEQ ID NO: 141 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99 % or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%) with SEQ ID NO: 135 , 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a miR-30 sequence leader sequence, which has the sequence of SEQ ID NO: 19; a miR-30 stem-loop sequence, which has the sequence of SEQ ID NO: The sequence of 4; and the miR-30 follower sequence, which has the sequence of SEQ ID NO: 34; and (b) the miR-218-1 leader sequence, which has the sequence of SEQ ID NO: 141; the miR-218-1 stem-loop The sequence, which has the sequence of SEQ ID NO: 135; and the miR-218-1 satellite sequence, which has the sequence of SEQ ID NO: 147. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 258 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 258.

In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) an hSyn promoter sequence that is at least 85% identical to any of the following (e.g., at least 85%, 90%, 95% , 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 194 to SEQ ID NO: 198, (b) miR-30 sequence guide sequence, which is identical to SEQ ID NO: 19 has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; miR-30 A stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence with SEQ ID NO: 4 Identity; and a miR-218-30 satellite sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO: 34 (e.g., 100%)) identity; and (c) a miR-218-1 guide sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97) identical to SEQ ID NO: 141 %, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%) with SEQ ID NO: 135 , 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that has at least 85% ( For example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) an hSyn promoter sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity, (b) a miR-30 sequence guide sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; sequence of 4; and miR-30 follower sequence , which is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to SEQ ID NO: 34; and (c) A miR-218-1 leader sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more () identical to SEQ ID NO: 141 For example, 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98) with SEQ ID NO: 135 %, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that has at least 85% (e.g., at least 85%, 90%, 95%) with SEQ ID NO: 147 , 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) hSyn promoter sequence, which has the sequence of SEQ ID NO: 198; (b) miR-30 sequence guide sequence, which has the sequence of SEQ ID NO : the sequence of 19; the miR-30 stem-loop sequence, which has the sequence of SEQ ID NO: 4; and the miR-30 follower sequence, which has the sequence of SEQ ID NO: 34; and (c) the miR-218-1 guide sequence , which has the sequence of SEQ ID NO: 141; the miR-218-1 stem-loop sequence, which has the sequence of SEQ ID NO: 135; and the miR-218-1 follower sequence, which has the sequence of SEQ ID NO: 147. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 259 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 259.

In some embodiments of any of the following nucleic acid molecules described herein, the nucleic acid molecule can include a single promoter that can control the expression of one or more (e.g., two) miRNA sequences, or two promoters that can each control the expression of one or more (e.g., two) miRNA sequences. Controlling the performance of individual miRNA constructs. For example, in some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a promoter sequence; (b) a miRNA sequence, such as a miR-30 sequence, including a miR-30 guide sequence, a miR-30 stem loop sequence and the miR-30 follower sequence; (c) optionally, the second promoter sequence; and (d) the second miRNA sequence, such as the miR-218 sequence, including the miR-218-1 leader sequence, miR-218-1 stem Loop sequence and miR-218-1 follower sequence. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a promoter sequence; (b) a miRNA sequence, such as a miR-30 sequence, including a miR-30 leader sequence, a miR-30 stem-loop sequence, and a miR-30 follower sequence; and (c) a second miRNA sequence, such as a miR-218 sequence, including a miR-218-1 leader sequence, a miR-218-1 stem-loop sequence, and a miR-218-1 follower sequence. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a promoter sequence; (b) a miRNA sequence, such as a miR-30 sequence, including a miR-30 leader sequence, a miR-30 stem-loop sequence, and The miR-30 follower sequence; (c) the second promoter sequence; and (d) the second miRNA sequence, such as the miR-218 sequence, including the miR-218-1 leader sequence, the miR-218-1 stem-loop sequence and the miR- 218-1 Follower sequence.

In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) an hSyn promoter sequence that is at least 85% identical to any of the following (e.g., at least 85%, 90%, 95% , 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 194 to SEQ ID NO: 198, (b) miR-30 sequence guide sequence, which is identical to SEQ ID NO: 19 has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; miR-30 A stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence with SEQ ID NO: 4 Identity; a sequence of 4; and a miR-30 follower sequence that is at least 85% identical to SEQ ID NO: 34 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identity; (c) a miR-218-1 guide sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%) with SEQ ID NO: 135 %, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that is at least 85% identical to SEQ ID NO: 147 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; and (d) Rabbit beta-globin (RBG) The polyadenylation (polyA) message sequence has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 213, SEQ ID NO: 214, and SEQ ID NO: 215. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) an hSyn promoter sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity, (b) a miR-30 sequence guide sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; sequence of 4; and miR-30 follower sequence , which is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to SEQ ID NO: 34; ( c) A miR-218-1 leader sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%, or more) identical to SEQ ID NO: 141 , 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%) with SEQ ID NO: 135 , 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and (d) the RBG polyadenylation message sequence is identical to one or more of the following (e.g., both , three, four or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity Properties: SEQ ID NO: 213, SEQ ID NO: 214 and SEQ ID NO: 215. In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) hSyn promoter sequence, which has the sequence of SEQ ID NO: 198; (b) miR-30 sequence guide sequence, which has the sequence of SEQ ID NO : the sequence of 19; the miR-30 stem-loop sequence, which has the sequence of SEQ ID NO: 4; and the miR-30 follower sequence, which has the sequence of SEQ ID NO: 34; (c) the miR-218-1 guide sequence, It has the sequence of SEQ ID NO: 141; the miR-218-1 stem-loop sequence, which has the sequence of SEQ ID NO: 135; and the miR-218-1 follower sequence, which has the sequence of SEQ ID NO: 147; and ( d) RBG polyadenylation message sequence having the sequence of any of the following: SEQ ID NO: 213, SEQ ID NO: 214 and SEQ ID NO: 215. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 260 or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 260.

In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a 5' ITR sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity, (b) an hSyn promoter sequence that has at least 85% (e.g., at least 85%) identity with SEQ ID NO: 198 , 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; (c) a miR-30 sequence guide sequence having SEQ ID NO: 19 At least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence that is SEQ ID NO: 4 has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; the sequence of 4 ; and a miR-30 satellite sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100) identical to SEQ ID NO: 34 %)) identity; (d) a miR-218-1 guide sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96) with SEQ ID NO: 135 %, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that is at least 85% (e.g., at least 85%) identical to SEQ ID NO: 147 , 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; (e) the RBG polyadenylation message sequence has one or more of the following (e.g., two, three, four, or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 213, SEQ ID NO: 214 and SEQ ID NO: 215; and (f), a 3' ITR sequence that is at least 85% identical to SEQ ID NO: 212 (e.g., At least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: 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 sequence; (c) a miR-30 sequence guide sequence that is at least 85% identical to SEQ ID NO: 19 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence having the sequence of SEQ ID NO: 4; and a miR-30 follower sequence having at least 85% (e.g., At least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity; (d) a miR-218-1 guide sequence that is identical to SEQ ID NO. : 141 Having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; miR-218-1 stem Loop sequence, which has the sequence of SEQ ID NO: 135; and miR-218-1 follower sequence, which has the sequence of SEQ ID NO: 147; (e) RBG polyadenylation message sequence and one or more of the following (e.g., two, three, four, or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 213, SEQ ID NO: 214 and SEQ ID NO: 215; and (f) a 3' ITR sequence having the sequence of SEQ ID NO: 212. In some embodiments, the nucleic acid molecule includes a nucleic acid sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 261 or more (eg, 100%)) sequence 261. In some embodiments, the nucleic acid molecule includes the nucleic acid sequence of SEQ ID NO: 261.

In some embodiments, the nucleic acid molecule includes: from 5' to 3', (a) a 5' ITR sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity, (b) an hSyn promoter sequence that has at least 85% (e.g., at least 85%) identity with SEQ ID NO: 198 , 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; sequence of 198; (c) miR-30 sequence guide sequence, which is identical to SEQ ID NO: 19 has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; miR-30 stem-loop A sequence that 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: 4 ; the sequence of 4; and a miR-30 follower sequence that is at least 85% identical to SEQ ID NO: 34 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity; (d) a miR-218-1 guide sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) identical to SEQ ID NO: 141 , 98%, 99% or more (e.g., 100%)) sequence identity; a miR-218-1 stem-loop sequence that has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and a miR-218-1 follower sequence that is at least 85% (e.g., 147) identical to SEQ ID NO: 147 , at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; (e) the RBG polyadenylation message sequence is identical to one of the following One or more (e.g., two, three, four, or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity: SEQ ID NO: 213, SEQ ID NO: 214, and SEQ ID NO: 215; and (f) a filler sequence that is identical to one or more of the following (e.g., Two, three, four, or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 250 and SEQ ID NO: 251; and (g) a 3' ITR sequence that is at least 85% (e.g., at least 85%, 90%, 95%, 96) identical to SEQ ID NO: 212 %, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, the nucleic acid molecule includes: 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 sequence; (c) a miR-30 sequence guide sequence that is at least 85% identical to SEQ ID NO: 19 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity; a miR-30 stem-loop sequence having the sequence of SEQ ID NO: 4; and a miR-30 follower sequence having at least 85% (e.g., At least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity; (d) a miR-218-1 guide sequence that is identical to SEQ ID NO. : 141 Having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity; miR-218-1 stem Loop sequence, which has the sequence of SEQ ID NO: 135; and miR-218-1 follower sequence, which has the sequence of SEQ ID NO: 147; (e) RBG polyadenylation message sequence and one or more of the following (e.g., two, three, four, or five) have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 213, SEQ ID NO: 214 and SEQ ID NO: 215; (f) filler sequence, which has one or more of the following (e.g., two, three , four or five): SEQ ID NO: 250 and SEQ ID NO: 251; and (g) 3' ITR sequence, which has the sequence of SEQ ID NO: 212.

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

Inhibitory nucleic acid molecules (eg, inhibitory RNA agents) of the present disclosure can be GluK2 inhibitors. Specifically, GluK2 inhibitors can be inhibitors of Grik2 mRNA expression. Inhibiting the expression of GluK2 can also inhibit GluK5 levels (Ruiz et al., J Neuroscience 2005). While not wishing to be bound by any theory, this disclosure is based on the principle that sufficient removal of GluK2 alone should remove all GluK2/GluK5 heteropolymers, since individual GluK5 subunits cannot form homopolymer assemblies.

According to the disclosed methods and compositions, the inhibitory nucleic acid molecules (e.g., inhibitory RNA agents) disclosed herein can be 15 to 50 nucleotides in length (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, inhibitory nucleic acid molecules (e.g., inhibitory RNA agents) disclosed herein may be 15 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 16 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 17 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 18 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 19 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 20 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 21 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 22 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 23 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 24 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 25 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 25 to 30 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 30 to 35 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 35 to 40 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 40 to 45 nucleotides in length. In another example, an inhibitory nucleic acid molecule (e.g., inhibitory RNA agent) is 45 to 50 nucleotides in length.

Inhibitory RNA agents of the present disclosure include sequences that are at least substantially complementary or completely complementary to a sequence region of Grik2 mRNA (e.g., any one of SEQ ID NO: 164 to SEQ ID NO: 174) or a variant thereof, which complement Sufficient to produce specific binding under intracellular conditions. In some embodiments, the inhibitory RNA agent includes a sequence that is at least substantially complementary or completely complementary to a sequence region of Grik2 mRNA, such as SEQ ID NO: 164, or that is at least 85% sequence identical to SEQ ID NO: 164 Variations of identity. For example, the present disclosure contemplates an inhibitory RNA agent having an antisense sequence that is identical to at least 7 (e.g., at least 7, 8, 9, 10, 11, 12, 13) of one or more regions of Grik2 mRNA. , 14, 15, 16, 17, 18, 19, 20, 21, 22 or more) consecutive nucleotide complements. In a specific example, the inhibitory RNA agent has an antisense sequence that is complementary to 7 contiguous nucleotides in one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 8 contiguous nucleotides in one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 9 contiguous nucleotides in one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 10 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 11 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 12 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 13 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 14 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 15 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 16 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 17 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 18 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 19 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 20 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is complementary to 21 contiguous nucleotides of one or more regions of Grik2 mRNA. In another example, the inhibitory RNA agent has an antisense sequence that is 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 nucleotides in one or more regions of Grik2 mRNA.

The present disclosure contemplates an inhibitory RNA agent that binds to one or more regions of Grik2 mRNA (eg, any of the regions of Grik2 mRNA described below: SEQ ID NO: 164 to SEQ ID NO: 174) When combined with Grik2 mRNA, 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 are formed duplex structure between acids. In some embodiments, the inhibitory RNA agent of the present disclosure can bind to the region of Grik2 mRNA within the sequence of SEQ ID NO: 164 and form with Grik2 mRNA a length of 7 to 22 (e.g., 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22) duplex structure between nucleotides. For example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 7 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 8 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 9 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 10 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 11 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 12 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 13 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 14 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 15 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 16 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 17 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 18 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 19 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 20 nucleotides in length. In another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 21 nucleotides in length. In yet another example, the duplex structure between the inhibitory RNA agent and Grik2 mRNA can be 10 nucleotides in length.

According to the disclosed methods and compositions, an inhibitory RNA agent (e.g., having at least 85% (at least 86%, 87%, 88%, 89%, 90%, 91%) a nucleic acid sequence with any of the following , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19. SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229 and SEQ ID NO: 238 to SEQ ID NO: 241), such as one of an inhibitory RNA agent (which has at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 258) and one of Grik2 mRNA or The duplex structure formed by the multiple regions may include at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) mismatches . For example, a duplex structure can contain 1 mismatch. In another example, the duplex structure contains 2 mismatches. In another example, the duplex structure contains 3 mismatches. In another example, the duplex structure contains 4 mismatches. In another example, the duplex structure contains 5 mismatches. In another example, the duplex structure contains 6 mismatches. In another example, the duplex structure contains 7 mismatches. In another example, the duplex structure contains 8 mismatches. In another example, the duplex structure contains 9 mismatches. In another example, the duplex structure contains 10 mismatches. In another example, the duplex structure contains 11 mismatches. In another example, the duplex structure contains 12 mismatches. In another example, the duplex structure contains 13 mismatches. In another example, the duplex structure contains 14 mismatches. In yet another example, the duplex structure contains 15 mismatches.

Accordingly, one object of the present disclosure relates to isolated synthetic or recombinant inhibitory nucleic acid molecules (eg, inhibitory RNA agents) that target Grik2 mRNA. 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 Grik2 expression inhibitors that are an inhibitory RNA agent (eg, siRNA, shRNA, miRNA, or shmiRNA). Inhibitory RNA agents, including antisense RNA molecules and antisense DNA molecules, can directly block its translation by binding to Grik2 mRNA and preventing protein translation or increasing mRNA degradation, thereby reducing the level and activity of GluK2 protein. For example, an inhibitory RNA agent that has at least about 19 bases and is complementary to a unique region of the mRNA transcript sequence encoding GluK2 can be synthesized, e.g., by conventional techniques (e.g., the techniques disclosed herein) and administered, e.g., by intravenous injection or Administration is by infusion, as well as other routes described herein, such as direct injection into areas of the brain. Methods for using antisense technology to specifically mitigate gene expression of genes whose sequence is known are known in the art (see, for example, U.S. Patent 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 All methods are incorporated into this article).

In specific examples, Grik2 inhibitory RNA agents of the present disclosure can be short interfering RNA (siRNA). Grik2 gene expression can be reduced by contacting individuals or cells with small double-stranded RNA (dsRNA) or vectors encoding it, resulting in degradation of the mRNA in a sequence-specific manner (e.g., via the RNA interference pathway ) to specifically inhibit the production of small double-stranded RNA expressed by Grik2 . Methods for selecting appropriate dsRNA or dsRNA-encoding vectors for genes whose sequences are known are known in the art (see, for example, Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619 and WO 01/68836 , each of which is incorporated herein by reference in its entirety).

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

In addition, the Grik2 expression inhibitor of the present disclosure may be microRNA (miRNA). miRNA has a general meaning in the art and refers to, for example, microRNA molecules that are typically 21 to 22 nucleotides in length, but lengths of 19 and up to 23 nucleotides have been reported and can be used to inhibit targeted Translation of its mRNA. Each of the miRNAs is processed from a longer precursor RNA molecule ("precursor-miRNA"). Precursor miRNAs are transcribed from non-protein-coding genes. Precursor miRNAs possess two complementary regions that enable them to form stem-loop- or fold-back-like structures that are cleaved in animals by a ribonuclease III-like nuclease called Dicer. Processed miRNA is usually part of a stem containing a "seed sequence" (usually 6 to 8 nucleotides) that is completely or substantially complementary to a region of the target mRNA. Processed miRNAs (also called "mature miRNAs") become part of large complexes to downregulate (eg, reduce translation or degrade the mRNA) specific target genes.

In addition, the GluK2 inhibitor of the present disclosure can be a miRNA-adapted shRNA (shmiRNA). shmiRNA agents refer to chimeric molecules that incorporate antisense sequences within the -5p or -3p arm of a microRNA scaffold (e.g., E-miR-30 scaffold) containing microRNA flanking and loop sequences. Compared to shRNA, shmiRNA usually has a longer stem-loop structure based on microRNA-derived sequences, in which the -5p and -3p arms exhibit complete or substantial complementarity (e.g., mismatch, G:U swing). Due to their long sequence and processing requirements, shmiRNAs are usually expressed from the Pol II promoter. These constructs also showed reduced toxicity compared to shRNA-based agents.

Various miRNAs can be used to knock down Grik2 mRNA expression (and subsequently its gene product GluK2). MiRNAs can be complementary to different target transcripts or to different binding sites on a single target transcript. Polycistronic or multigene transcripts can also be used to increase the efficiency of target gene knockdown. Multiple genes encoding the same miRNA 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 within 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.

Inhibitory RNAs that are complementary (eg, substantially or completely complementary) to the sense target sequence of Grik2 mRNA are typically encoded by the DNA sequence used to generate any of the aforementioned inhibitors (eg, siRNA, shRNA, miRNA, or shmiRNA). DNA encoding the double-stranded RNA of interest can be incorporated into a genetic cassette (eg, an expression cassette in which transcription of the DNA is controlled by a promoter). Enhanced RISC loading of guide sequences

One step in RNA interference involves the assembly of microRNA guide strands into the RNA-induced silencing complex (RISC) protein complex, which mediates target mRNA cleavage. MicroRNAs are produced as double-stranded duplexes containing a leader strand that hybridizes by complementary base pairing with a follower strand. Assembly of the leader strand into the RISC complex is often accompanied by degradation of the follower strand. RISC assembly favors microRNA strands with 5' ends that are more likely to fray or be released from the duplex. The constructs described herein are designed by destabilizing base pairing at the 5' end of the guide strand (e.g., by introducing a U-A pair or a U-G rocker pair at or near the 5' end of the guide strand) and causing Base pairing tightening at the 5' end of the follower strand (e.g., by introducing G-C pairs at or near the 5' end of the follower strand) to facilitate guide selection and loading by RISC and to the detriment of follower selection . This strategy is possible if the mismatch between the guide strand and the target mRNA occurs at the first nucleotide of the guide strand or close to the 3' end (e.g., within the last four nucleotides) , they are well tolerated. Such strategies not only enhance on-target knockdown of the guide strand but also reduce off-target effects caused by the generation or retention of the follower strand by the RISC protein complex.

Accordingly, anti -Grik2 antisense molecules (e.g., microRNA, shRNA, siRNA, or shmiRNA) described herein include one or more modifications that enhance RISC loading or retention of the leader strand and reduce RISC loading or retention of the follower strand. , increasing the ratio of intracellular leader strands to follower strands and increasing the level of knockdown of the target Grik2 mRNA.

In several of the constructs described here, base pairing instability is increased at or near the 5' end of the guide strand to enhance RISC loading or retention of the guide strand. For example, base pairing instability was achieved by introducing U-A pairs or U-G wobble pairs at or near the 5' end of the leading strand of several constructs.

In several constructs described herein, RISC loading or retention of the follower strand is reduced by introducing base pairing instability at the 5' end of the follower strand. Base pairing instability is introduced by adding C-G pairs at or near the 5' end of the follower strand.

Several constructs of the present disclosure are also designed to enhance RISC loading or retention of the guide strand by introducing a 5'-terminal uracil into the guide strand. This 5' terminal nucleotide is not involved in hybridization to the target mRNA (eg, Grik2 mRNA) and is normally anchored in the phosphate binding pocket of the Argonaute RISC catalytic component 2 (Ago2) protein.

By introducing one or more mismatches (e.g., 1, 2, 3, 4, 5, 6, 7, or (more mismatches), the RISC loading or retention of the follower strands of several of the disclosed constructs was reduced. This strategy is used to facilitate the unfolding and unloading of follower strands during RISC loading. Although extensive complementarity in the seed region of the guide strand (guide nucleotides 2 to 8, g2 to g8) and the middle region is critical for Ago2-mediated mRNA cleavage, base pairing at the 3' end is not required. Indeed, it was determined that mismatches of the guide strand at positions g18, g19, g20, and g21 with the target mRNA attenuate the release of the guide strand from Ago2, an unloading activity mediated by the target mRNA.

For several of the constructs disclosed herein, Dicer cleavage that excises the loop region from the stem-loop structure of the Grik2 construct is enhanced by replacing the UG rocker pairs with CG pairs. The Grik2 mRNA targeting constructs of the present disclosure utilize the aforementioned modifications to promote an increase in the ratio of leader to follower strand production and enhance silencing of Grik2 to treat epileptic seizure disorders (eg, TLE).

Accordingly, inhibitory RNA molecules described herein may include a stem-loop sequence containing anti- Grik2 sequence GI (SEQ ID NO: 16) embedded within an E-miR-30 microRNA scaffold and a sequence complementary thereto ( See, for example, Table 2 (e.g., SEQ ID NO: 1 to SEQ ID NO: 15, SEQ ID NO: 226 to SEQ ID NO: 229, and SEQ ID NO: 238 to SEQ ID NO: 241)) or have at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Lead and follower strand sequences are rationally designed whose variants have multiple (e.g., 100%) sequence identity. Table 2 : Grik2 targeting constructs containing antisense sequence GI or variants thereof Antisense sequence * microRNA scaffold _ Version stem-loop sequence SEQ ID NO GI (GI; SEQ ID NO: 16) E-miR-30 Construct A gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac GTCTCGATATGGAGAACCCATG ctgtgaagccacagatggg catgggttttatatcgagac gctgcctactgcctcggaC ttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 1 GI E-miR-30 Construct #1 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G c C gtgaagccacagatggg c C C tgggttttatatcgaga A gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 2 GI E-miR-30 Construct #2 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G c C gtgaagccacagatggg c C C tgggttttatatcg C ga A gctgcctact gcctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 3 GI E-miR-30 Construct #3 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G c C gtgaagccacagatggg c C C tgggttttatatcgag CA gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 4 GI E-miR-30 Construct #4 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G c C gtgaagccacagatggg c C C tgggttttatatcga T a A gctgcctactg cctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 5 GI E-miR-30 Construct #5 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG c C gtgaagccacagatggg catgggttttatatcg C ga A gctgcctactgcctc ggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 6 GI E-miR-30 Construct #6 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG c C gtgaagccacagatggg catgggttttatatcgaga A gctgcctactgcctcgga Cttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 7 GI E-miR-30 Construct #7 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatggg c C C tgggttttatatcgaga A gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 8 GI E-miR-30 Construct #8 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatggg c C C tgggttttatatcg C ga A gctgcctact gcctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 9 GI E-miR-30 Construct #9 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG ctgtgaagccacagatggg catgggttttatatcg C ga A gctgcctactgcctc ggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 10 GI E-miR-30 Construct #10 > Cttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta _ _ 11 GI E-miR-30 Construct #11 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatggg c C C tgggttttatatcg C ga G gctgcctact gcctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 12 GI E-miR-30 Construct #12 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatggg c C C tgggttttatatcgaga G gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 13 GI E-miR-30 Construct #13 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG ctgtgaagccacagatggg catgggttttatatcg C ga G gctgcctactgcctc ggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 14 GI E-miR-30 Construct #14 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG ctgtgaagccacagatggg catgggttttatatcgaga G gctgcctactgcctcgga Cttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 15 GI E-miR-30 Construct #15 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatg A gc C tgggttttatatcgaga A gctgcctactg cctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 226 GI E-miR-30 Construct #16 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G ctgtgaagccacagatg A gc C tgggttttatatcg C ga A gctgcct actgcctcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtataaatta 227 GI E-miR-30 Construct #17 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG ctgtgaagccacagatg A gcatgggttttatatcgaga A gctgcctactgcctcgg aCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 228 GI E-miR-30 Construct #18 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCATG ctgtgaagccacagatg A gcatgggttttatatcg C ga A gctgcctactgcct cggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 229 GI E-miR-30 Construct #19 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G cugtgaagccacagatggg c C C tgggttttatatcgag CA gctgcctactgcct cggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 238 GI E-miR-30 Construct #20 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G cugtgaagccacagatggg c C C tgggttttatatcgag CG gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 239 GI E-miR-30 Construct #21 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G G cugtgaagccacagatggg c C C tgggttttatatcgag GG gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 240 GI E-miR-30 Construct #22 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac T TTCCGATATGGAGAACCCA G cugtgaagccacagatggg c C C tgggttttatatcgag GA gctgcctactgcc tcggaCttcaaggggctactttaggagcaattatcttgtttactaaaactgaataccttgctatctctctttgatacatttttacaaagctgaattaaaatggtataaatta 241 Table 2 Sequence Key: * The term "antisense sequence" used in Table 2 refers to the antisense sequence GI or it having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Single and double underlined characters : stem-loop sequence; single-underlined uppercase italic characters : leader strand; single-underlined lowercase characters : E-miR-30 loop sequence; double-underlined characters : follower strand; uppercase bold Character : substituted nucleotide.

Accordingly, the Grik2 targeting antisense construct of the present disclosure may include the guide strands described in Table 3 below (SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) and the accompanying shares (SEQ ID NO: 31 to SEQ ID NO: 45, SEQ ID NO: 234 to SEQ ID NO: 237 and SEQ ID NO: 246 to SEQ ID NO: 249 ): Table 3 : Guide and follower pairs rationally designed from the GI sequence in the E-miR-30 scaffold Antisense sequence * microRNA scaffold _ Version boot sequence SEQ ID NO Follower sequence SEQ ID NO GI E-miR-30 Construct A gtctcgatatggagaacccatg 16 catgggttttatatcgagac 31 GI E-miR-30 Construct #1 T tctcgatatggagaaccca G g 17 c C tgggttttatatcgaga A 32 GI E-miR-30 Construct #2 T tctcgatatggagaaccca G g 18 c C tgggttttatatcg C ga A 33 GI E-miR-30 Construct #3 T tctcgatatggagaaccca G g 19 c C tgggttttatatcgag CA 34 GI E-miR-30 Construct #4 T tctcgatatggagaaccca G g 20 c C tgggttttatatcga T a A 35 GI E-miR-30 Construct #5 T tctcgatatggagaacccatg twenty one catgggttttatatcg C ga A 36 GI E-miR-30 Construct #6 T tctcgatatggagaacccatg twenty two catgggttttatatcgaga A 37 GI E-miR-30 Construct #7 T tctcgatatggagaaccca G g twenty three c C tgggttttatatcgaga A 38 GI E-miR-30 Construct #8 T tctcgatatggagaaccca G g twenty four c C tgggttttatatcg C ga A 39 GI E-miR-30 Construct #9 T tctcgatatggagaacccatg 25 catgggttttatatcg C ga A 40 GI E-miR-30 Construct #10 T tctcgatatggagaacccatg 26 catgggttttatatcgaga A 41 GI E-miR-30 Construct #11 T tctcgatatggagaaccca G g 27 c C tgggttttatatcg C ga G 42 GI E-miR-30 Construct #12 T tctcgatatggagaaccca G g 28 c C tgggttttatatcgaga G 43 GI E-miR-30 Construct #13 T tctcgatatggagaacccatg 29 catgggttttatatcg C ga G 44 GI E-miR-30 Construct #14 T tctcgatatggagaacccatg 30 catgggttttatatcgaga G 45 GI E-miR-30 Construct #15 T tctcgatatggagaaccca G g 230 A gc C tgggttttatatcgaga A 234 GI E-miR-30 Construct #16 T tctcgatatggagaaccca G g 231 A gc C tgggttttatatcg C ga A 235 GI E-miR-30 Construct #17 T tctcgatatggagaacccatg 232 A gcatgggttttatatcgaga A 236 GI E-miR-30 Construct #18 T tctcgatatggagaacccatg 233 A gcatgggttttatatcg C ga A 237 GI E-miR-30 Construct #19 T tctcgatatggagaaccca G g 242 c C tgggttttatatcgag CA 246 GI E-miR-30 Construct #20 T tctcgatatggagaaccca G g 243 c C tgggttttatcgag CG 247 GI E-miR-30 Construct #21 T tctcgatatggagaaccca G g 244 c C tgggttttatatcgag GG 248 GI E-miR-30 Construct #22 T tctcgatatggagaaccca G g 245 c C tgggttttatatcgag GA 249 Table 3 Sequence Key: * The term "antisense sequence" used in Table 3 refers to the antisense sequence GI or it having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Capital bold characters : nucleotides in the modified leader or follower sequence relative to construct A.

Also disclosed herein are inhibitory RNA molecules that may include a stem-loop sequence containing anti -Grik2 sequence G9 (SEQ ID NO: 63) embedded within the E-miR-124-3 microRNA scaffold and a sequence complementary thereto (See, e.g., Table 4 (e.g., SEQ ID NO: 46 to SEQ ID NO: 62)) or at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) of sequence identity for rationally designed lead and follower variants thereof stock sequence. Table 4 : Grik2 targeting constructs containing antisense sequence G9 or variants thereof Antisense sequence* microRNA scaffold Version stem-loop sequence SEQ ID NO G9 (SEQ ID NO: 63) E-miR-124-3 Construct B tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc tacaatgggcactagacatggg atttaatgtctatacaat CCCATAGCTAATGCCTGTTTTA gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggccccggcggccattcgcgccgcggacaa atccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 46 G9 E-miR-124-3 Construct #23 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc t G caa CA ggcactagacatggg atttaatgtctatacaat T CCATAGCTAATGCCTGTT GC A gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaggaaggcgagcccggccccggcggccattcgcgccgc ggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 47 G9 E-miR-124-3 Construct #24 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc t G caa cA ggcactagacatgg atttaatgtctatacaat T CATAGCTAATGCCTGTT GC A gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaaaggcgagcccggccccggcggccattcgcgccgc ggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 48 G9 E-miR-124-3 Construct #25 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc t G caatgggcactagacatggg atttaatgtctatacaat T CCATAGCTAATGCCTGTT GC A gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgccgcgg acaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 49 G9 E-miR-124-3 Construct #26 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc tacaa CA ggcactagacatggg atttaatgtctatacaat T CCATAGCTAATGCCTGTT G TA gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgccgcgg acaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 50 G9 E-miR-124-3 Construct #27 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc tacaatgggcactagacatggg atttaatgtctatacaat T CCATAGCTAATGCCTGTT G TA gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaaaggcgagcccggcccccggcggccattcgcgccgcggac aaatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 51 G9 E-miR-124-3 Construct #28 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc t G caatgggcactagacatgg atttaatgtctatacaat T CATAGCTAATGCCTGTT GC A gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaaaggcgagcccggcccccggcggccattcgcgccgcggac aaatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 52 G9 E-miR-124-3 Construct #29 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc tacaa CA ggcactagacatgg atttaatgtctatacaat T CATAGCTAATGCCTGTT G TA gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgccgcggac aaatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 53 G9 E-miR-124-3 Construct #30 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc tacaatgggcactagacatgg atttaatgtctatacaat T CATAGCTAATGCCTGTT G TA gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaggaaggcgagcccggcccccggcggccattcgcgccgcggaca aatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 54 G9 E-miR-124-3 Construct #31 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT cccatagctaatgcctgt GCG a ttaatgtctataca tcgcacaggcatcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgc gccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 55 G9 E-miR-124-3 Construct #32 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT T ccatagctaatgcctgt GCG attaatgtctataca tcgcacaggcatcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcg ccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 56 G9 E-miR-124-3 Construct #33 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT cccatagctaatgcctgt GCG A ttaatgtctataca tcgcaca TT catcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcg cgccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 57 G9 E-miR-124-3 Construct #34 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT T ccatagctaatgcctgt GCG A ttaatgtctataca tcgcaca TT catcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcg cgccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 58 G9 E-miR-124-3 Construct #35 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT cccatagctaatgcctgtttta ttaatgtctataca taaaacaggcatcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcg ccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 59 G9 E-miR-124-3 Construct #36 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT T ccatagctaatgcctgtttta ttaatgtctataca taaaacaggcatcagctatggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcg ccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 60 G9 E-miR-124-3 Construct #37 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT cccatagctaatgcctgt GCG a ttaatgtctataca t CGC acaggcatcagct C tggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattc gcgccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 61 G9 E-miR-124-3 Construct #38 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc TT T ccatagctaatgcctgt GCG a ttaatgtctatacat CGC acaggcatcagct C tggtaa gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgc gccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 62 Table 4 Sequence Key: * The term "antisense sequence" used in Table 4 refers to the antisense sequence G9 or it having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Single and double underlined characters : stem-loop sequence; single-underlined uppercase italic characters : leading strand; single-underlined lowercase characters : E-miR-124-3 loop sequence; double-underlined characters : follower strand; uppercase Bold characters : substituted nucleotides.

Accordingly, the Grik2- targeting antisense construct of the present disclosure may include the leader strand (SEQ ID NO: 63 to SEQ ID NO: 79) and the follower strand (SEQ ID NO: 80 to SEQ ID NO) as described in Table 5 below. : 96) Pair: Table 5 : Rationally designed leader and follower strand pairs from the G9 sequence in the miR-124 scaffold Antisense sequence * miRNA scaffold Version boot sequence SEQ ID NO Follower sequence SEQ ID NO G9 E-miR-124-3 Construct B cccatagctaatgcctgtttta 63 tacaatgggcactagacatggg 80 G9 E-miR-124-3 Construct #23 T ccatagctaatgcctgtt GC a 64 t G caa CA ggcactagacatggg 81 G9 E-miR-124-3 Construct #24 T catagctaatgcctgtt GC a 65 t G caa CA ggcactagacatgg 82 G9 E-miR-124-3 Construct #25 T ccatagctaatgcctgtt GC a 66 t G caatgggcactagacatggg 83 G9 E-miR-124-3 Construct #26 T ccatagctaatgcctgtt G ta 67 tacaa CA ggcactagacatggg 84 G9 E-miR-124-3 Construct #27 T ccatagctaatgcctgtt G ta 68 tacaatgggcactagacatggg 85 G9 E-miR-124-3 Construct #28 T catagctaatgcctgtt GC a 69 t G caatgggcactagacatgg 86 G9 E-miR-124-3 Construct #29 T catagctaatgcctgtt G ta 70 tacaa CA ggcactagacatgg 87 G9 E-miR-124-3 Construct #30 T catagctaatgcctgtt G ta 71 tacaatgggcactagacatgg 88 G9 E-miR-124-3 Construct #31 cccatagctaatgcctgt GCG a 72 tcgcacaggcatcagctatggtaa 89 G9 E-miR-124-3 Construct #32 T ccatagctaatgcctgt GCG a 73 tcgcacaggcatcagctatggtaa 90 G9 E-miR-124-3 Construct #33 cccatagctaatgcctgt GCG a 74 tcgcaca TT catcagctatggtaa 91 G9 E-miR-124-3 Construct #34 T ccatagctaatgcctgt GCG a 75 tcgcaca TT catcagctatggtaa 92 G9 E-miR-124-3 Construct #35 cccatagctaatgcctgtttta 76 taaaacaggcatcagctatggtaa 93 G9 E-miR-124-3 Construct #36 T ccatagctaatgcctgtttta 77 taaaacaggcatcagctatggtaa 94 G9 E-miR-124-3 Construct #37 cccatagctaatgcctgt GCG a 78 t CGC acaggcatcagct C tggtaa 95 G9 E-miR-124-3 Construct #38 T ccatagctaatgcctgt GCG a 79 t CGC acaggcatcagct C tggtaa 96 Table 5 Sequence Key: * The term "antisense sequence" used in Table 5 refers to the antisense sequence G9 or it having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Capital bold characters : nucleotides in the modified leader or follower sequence relative to construct B.

Inhibitory RNA molecules described herein may include a stem-loop sequence containing anti -Grik2 sequence MW (SEQ ID NO: 109) embedded within the E-miR-124-3 microRNA scaffold and a sequence complementary thereto ( See, e.g., Table 6 (e.g., SEQ ID NO: 97 to SEQ ID NO: 108)) or at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity and its variants are reasonably designed to lead and follower strands sequence. Table 6 : Grik2 targeting constructs containing antisense sequence MW or variants thereof Antisense sequence * miRNA scaffold Version stem-loop sequence SEQ ID NO MW (SEQ ID NO: 109) E-miR-124-3 Construct C tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc gacgtttatctacaacactctg atttaatgtctatacaat CAGAGCATTGCAGATGGACTGC gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggccccggcggccattcgcgccgcggacaa atccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 97 MW E-miR-124-3 Construct #39 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc g CA gt CC atctacaacactctg atttaatgtctatacaat T AGAGCATTGCAGATGGACTGC gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgccgcggaca aatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 98 MW E-miR-124-3 Construct #40 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga G gtt C atctacaacact T t A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcg ccgcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 99 MW E-miR-124-3 Construct #41 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga G gt CC atctacaacact T t A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgcc gcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 100 MW E-miR-124-3 Construct #42 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc g CA gtttatctacaacactctg atttaatgtctatacaat T AGAGCATTGCAGATGGACTGC gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaggaaggcgagcccggcccccggcggccattcgcgccgcggaca aatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 101 MW E-miR-124-3 Construct #43 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga A gt CC atctacaacactctg atttaatgtctatacaat T AGAGCATTGCAGATGGACTGC gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaaaggcgagcccggcccccggcggccattcgcgccgcggac aaatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 102 MW E-miR-124-3 Construct #44 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga A gtttatctacaacactctg atttaatgtctatacaat T AGAGCATTGCAGATGGACTGC gagaggcgcctccgccgctccttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggagaaggcgagcccggcccccggcggccattcgcgccgcggac aaatccggcgaacaatgcgcccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 103 MW E-miR-124-3 Construct #45 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga A gtttatctacaacactct A atttaatgtctatacaat T AGAGCATTGCAGATGGACTGCg agaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgccgcgg acaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 104 MW E-miR-124-3 Construct #46 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga G gtt C atctacaacactct A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggccccggcggccattcgcgccg cggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 105 MW E-miR-124-3 Construct #47 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga G gtttatctacaacactct A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaggaaggcgagcccggccccggcggccattcgcgccgc ggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 106 MW E-miR-124-3 Construct #48 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc ga G gt CC atctacaacactct A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcggggaggaaggcgagcccggccccggcggccattcgcgccgc ggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 107 MW E-miR-124-3 Construct #49 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc g CG gt CC atctacaacact T t A atttaatgtctatacaat T AGAGCATTGCAGATGGAC C GC gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccggcccccggcggccattcgcgcc gcggacaaatccggcgaacaatgcgcccgcccagtgcggcccagctgccgggccggggatctggccgcgggacacaaaggggcccgcacgcctctggcgt 108 Table 6 Sequence Key: * The term "antisense sequence" used in Table 6 refers to an antisense sequence MW or one having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Single and double underlined characters : stem-loop sequence; single-underlined uppercase italic characters : leading strand; single-underlined lowercase characters : E-miR-124-3 loop sequence; double-underlined characters : follower strand; uppercase Bold characters : substituted nucleotides.

Accordingly, the Grik2- targeting antisense construct of the present disclosure may include the leader strand (SEQ ID NO: 109 to SEQ ID NO: 120) and the follower strand (SEQ ID NO: 121 to SEQ ID NO) as described in Table 7 below. : 132) Pair: Table 7 : Rationally designed leader and follower pairs from the MW sequence in the miR-124 scaffold Antisense sequence * miRNA scaffold Version boot sequence SEQ ID NO Follower sequence SEQ ID NO MW E-miR-124-3 Construct C cagagcattgcagatggactgc 109 gacgtttatctacaacactctg 121 MW E-miR-124-3 Construct #39 T agagcattgcagatggactgc 110 g CA gt CC atctacaacactctg 122 MW E-miR-124-3 Construct #40 T agagcattgcagatggac C gc 111 ga G gtt C atctacaacact T t A 123 MW E-miR-124-3 Construct #41 T agagcattgcagatggac C gc 112 ga G gt CC atctacaacact T t A 124 MW E-miR-124-3 Construct #42 T agagcattgcagatggactgc 113 g CA gtttatctacaacactctg 125 MW E-miR-124-3 Construct #43 T agagcattgcagatggactgc 114 ga A gt CC atctacaacactctg 126 MW E-miR-124-3 Construct #44 T agagcattgcagatggactgc 115 ga A gtttatctacaacactctg 127 MW E-miR-124-3 Construct #45 T agagcattgcagatggactgc 116 ga A gtttatctacaacactct A 128 MW E-miR-124-3 Construct #46 T agagcattgcagatggac C gc 117 ga G gtt C atctacaacactct A 129 MW E-miR-124-3 Construct #47 T agagcattgcagatggac C gc 118 ga G gtttatctacaacactct A 130 MW E-miR-124-3 Construct #48 T agagcattgcagatggac C gc 119 ga G gt CC atctacaacactct A 131 MW E-miR-124-3 Construct #49 T agagcattgcagatggac C gc 120 g CG gt CC atctacaacact T t A 132 Table 7 Sequence Key: * The term "antisense sequence" used in Table 7 refers to an antisense sequence MW or one having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Capital bold characters : nucleotides in the modified leader or follower sequence relative to construct C.

Inhibitory RNA molecules described herein may include a stem-loop sequence containing anti -Grik2 sequence MW (SEQ ID NO: 109) embedded within the E-miR-218 microRNA scaffold and sequences complementary thereto (see, For example, Table 8 (e.g., SEQ ID NO: 133 to SEQ ID NO: 138)) or has at least 85% of the %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity of the lead and follower strands. Table 8 : Grik2 targeting constructs containing antisense sequence MW or variants thereof Antisense sequence * miRNA scaffold Version stem-loop sequence SEQ ID NO MW E-miR-218-1 Construct D acgtttccagaacgtctgtagcttttctcctccttccccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg CAGAGCATTGCAGATGGACTG ggttgcgaggtatgagtaaa cagtccatacgcaatgctccg tggaacgtcacgcagctttctacagcat gacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 133 MW E-miR-218-1 Construct #50 acgtttccagaacgtctgtagcttttctcctccttccctccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg T AGAGCATTGCAGATGG C C C G ggttgcgaggtatgagtaaa c G g G ccatacgcaatgctcc A tggaacgtcacgcagcttt ctacagcatgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 134 MW E-miR-218-1 Construct #51 acgtttccagaacgtctgtagcttttctcctccttccccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg T AGAGCATTGCAGATGGAC C G ggttgcgaggtatgagtaaa c G gtccatacgcaatgctcc A tggaacgtcacgcagctttct acagcatgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 135 MW E-miR-218-1 Construct #52 acgtttccagaacgtctgtagcttttctcctccttccctccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg CAGAGCATTGCAGATGG C C G ggttgcgaggtatgagtaaa c G g G ccatacgcaatgctccg tggaacgtcacgcagctttct acagcatgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 136 MW E-miR-218-1 Construct #53 acgtttccagaacgtctgtagcttttctcctccttccccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg C AGAGCATTGCAGATGGAC C G ggttgcgaggtatgagtaaa c G gtccatacgcaatgctccg tggaacgtcacgcagctttct acagcatgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 137 MW E-miR-218-1 Construct #54 acgtttccagaacgtctgtagcttttctcctccttccccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg T AGAGCATTGCAGATGGACTG ggttgcgaggtatgagtaaa cagtccatacgcaatgctcc A tggaacgtcacgcagctttctacag catgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg 138 Table 8 Sequence Key: * The term "antisense sequence" used in Table 8 refers to an antisense sequence MW or one having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Single and double underlined characters : stem-loop sequence; single-underlined uppercase italic characters : leading strand; single-underlined lowercase characters : E-miR-218 loop sequence; double-underlined characters : follower strand; uppercase bold Character : substituted nucleotide.

Accordingly, the Grik2- targeting antisense construct of the present disclosure may include the leader strand (SEQ ID NO: 139 to SEQ ID NO: 144) and the follower strand (SEQ ID NO: 145 to SEQ ID NO) as described in Table 9 below : 146) Pair: Table 9 : Rationally designed guide and follower pairs from the MW sequence in the miR-218 scaffold Antisense sequence * miRNA scaffold Version boot sequence SEQ ID NO Follower sequence SEQ ID NO MW E-miR-218-1 Construct D cagagcattgcagatggactg 139 cagtccatacgcaatgctccg 145 MW E-miR-218-1 Construct #50 T agagcattgcagatgg C c C g 140 c G g G ccatacgcaatgctcc A 146 MW E-miR-218-1 Construct #51 T agagcattgcagatggac C g 141 c G gtccatacgcaatgctcc A 147 MW E-miR-218-1 Construct #52 cagagcattgcagatgg C c C g 142 c G g G ccatacgcaatgctccg 148 MW E-miR-218-1 Construct #53 cagagcattgcagatggac C g 143 c G gtccatacgcaatgctccg 149 MW E-miR-218-1 Construct #54 T agagcattgcagatggactg 144 cagtccatacgcaatgctcc A 150 Table 9 Sequence Key: * The term "antisense sequence" used in Table 9 refers to an antisense sequence MW or one having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) modifications ( For example, substitutions, deletions, insertions or mismatches). Capital bold characters : nucleotides in the modified leader or follower sequence relative to construct D.

The aforementioned sequences are represented as DNA (ie, cDNA) sequences that can be incorporated into the vectors of the present disclosure. These sequences can also be expressed as corresponding RNA sequences synthesized from vectors within the cell. One skilled in the art will understand that the cDNA sequence is equivalent to the mRNA sequence except that uridine is replaced by thymidine and can be used for the same purpose herein, namely, to generate polynucleotides for inhibiting the expression of Grik2 mRNA. In the case of DNA vectors (eg, AAV), the polynucleotide containing the antisense nucleic acid is the DNA sequence. In the case of RNA vectors, the gene transfer cassette incorporates the RNA equivalent of the antisense DNA sequence described herein.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 1 , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 1 Sequence identity. In another example, the inhibitory RNA can be at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%) identical to the nucleic acid sequence of SEQ ID NO: 1 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 1.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 2). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 2 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 2 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 2.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 3). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 3 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 3 ) sequence. Identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 3.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 4). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 4 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 4 ) sequence 4 identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 4.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 5 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 5 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 5.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 6). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 6 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 6 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 6.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 7). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 7 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 7 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 7.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 8). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 8 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 8 ) sequence. Identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 8.

The inhibitory RNA sequence of the present disclosure may have at least 85% (for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (for example) the nucleic acid sequence of SEQ ID NO: 9). , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 9 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 9 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 9.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 10 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 10 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 10.

The inhibitory RNA sequence of the present disclosure and the nucleic acid sequence of SEQ ID NO: 11 may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 11 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 11 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 11.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 12 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 12 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 12.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 13 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 13 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 13.

The inhibitory RNA sequence of the present disclosure and the nucleic acid sequence of SEQ ID NO: 14 may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 14 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 14 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 14.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 15 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 15 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 15.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 226 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 226 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 226.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 227 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 227 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 227.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 228 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 228 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 228.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 229 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 229 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 229.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 238 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 238 ) sequence. Identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 238.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 239 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 239 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 239.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 240 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 240 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 240.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 241 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 241 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 241.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 46 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 46 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 46.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 47 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 47 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 47.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 48 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 48 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 48.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 49 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 49 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 49.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 50 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 50 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 50.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 51 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 51 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 51.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 52 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 52 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 52.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 53 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 53 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 53.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 54 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 54 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 54.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 55 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 55 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 55.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 56 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 56 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 56.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 57 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 57 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 57.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% identical (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 58 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 58 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 58.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 59 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 59 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 59.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 60 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 60 ) Sequence In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 60.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 61 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 61 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 61.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 62 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 62 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 62.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 97 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 97 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 97.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 98 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 98 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 98.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 99 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 99 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 99.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 100 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 100 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 100.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 101 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 101 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 101.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 102 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 102 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 102.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 103 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 103 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 103.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 104 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 104 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 104.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 105 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 105 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 105.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 106 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 106 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 106.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 107 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 107 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 107.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 108 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 108 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 108.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 133 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 133 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 133.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 134 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 134 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 134.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 135 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 135 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 135.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 136 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 136 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 136.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 137 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 137 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 137.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 138 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 138 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 138.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 258 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 258 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 258.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 259 Sequence identity. In another example, the inhibitory RNA may be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 259 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 259.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 260 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 260 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 260.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 261 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 261 ) sequence identity. In yet another example, the inhibitory RNA can have the nucleic acid sequence of SEQ ID NO: 261.

The inhibitory RNA sequence of the present disclosure may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., at least 85%, 90%, 95%, 99%) or more (e.g. , 100%)) sequence identity. For example, the inhibitory RNA may be at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to the nucleic acid sequence of SEQ ID NO: 256 Sequence identity. In another example, the inhibitory RNA can be at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) to the nucleic acid sequence of SEQ ID NO: 256 ) sequence identity. In yet another example, the inhibitory RNA may have the nucleic acid sequence of SEQ ID NO: 256. Inhibitory polynucleotides with wobble base pairs

Further features of the present disclosure are inhibitory RNA agents having one or more wobble base pairs. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A) and hypoxanthine-cytosine (I-C). Xanthine represents the nucleoside inosine. The G-U wobble base pair has shown similar thermodynamic stability to G-C, A-T, and A-U (Saxena et al., 2003, J Biol Chem, 278(45):44312-9).

Accordingly, the present disclosure provides an inhibitory RNA agent having a nucleotide sequence that shares at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 164 to SEQ ID NO: 174 (e.g., between the inhibitory RNA and the Grik2 gene sequence Antisense shares may have 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). Specifically, inhibitory RNA agents of the present disclosure may have 1, 2, or 3 corresponding aligned human Grik2 mRNA transcripts ( For example, any of SEQ ID NO: 164 to SEQ ID NO: 174) are non-complementary nucleotides. Therefore, the inhibitory RNA agent of the present disclosure can have a nucleotide sequence that is consistent with the following: Any of the target regions has at least 85% complement (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)), at least 86 % (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)), at least 87% (e.g., at least 87%, 90%, 95 %, 96%, 97%, 98%, 99% or more (e.g., 100%)), at least 88% (e.g., at least 88%, 90%, 95%, 96%, 97%, 98%, 99 % or more (e.g., 100%)), at least 89% (e.g., at least 89%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) or At least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) identical to: SEQ ID NO: 164 to SEQ ID NO: 174. Identical to all Nucleotides where the complementary sequences of the aligned Grik2 mRNA sequences are not 100% identical may be wobble nucleotides.

The probability of off-target effects mediated by antisense RNA designed to target specific regions on the Grik2 transcript can be measured using any number of publicly available algorithms. For example, one can use the online tool siSPOTR ("SiRNA Sequence Off-Target Probability Reduction", available at world-wide-web.sispotr.icts.uiowa.edu/sispotr/index.html_).

Inhibitory RNA agents disclosed herein target a GluK2 protein encoding a GluK2 protein (eg, a GluK2 protein including any one of SEQ ID NO: 151 to SEQ ID NO: 163, or at least amino acid 1 including SEQ ID NO: 151 to 509 of GluK2 protein) mRNA. An mRNA encoding a GluK2 protein may include a polynucleotide encoding a polypeptide that contains one or more amino acid substitutions, such as one or more retaining amino acid substitutions, relative to a polypeptide having the sequence of any of the following (For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid substitutions such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more retained amino acid substitutions): SEQ ID NO: 151 to SEQ ID NO: 163. Grik2 protein and polynucleotide encoding it

Grik2 inhibitory RNA agents disclosed herein can be designed starting from the sequence of Grik2 mRNA using, for example, bioinformatics tools. The Grik2 mRNA sequence can be found at NCBI Gene ID NO: 2898. In another example, a polynucleotide sequence encoding SEQ ID NO: 151, a polynucleotide sequence encoding contiguous amino acids 1 to 509 of SEQ ID NO: 151, or an amino acid encoding any of the following Polynucleotide sequence of the sequence: SEQ ID NO: 151 (UniProtKB Q13002-1), SEQ ID NO: 152 (UniProtKB Q13002-2), SEQ ID NO: 153 (UniProtKB Q13002-3), SEQ ID NO: 154 (UniProtKB Q13002-4), SEQ ID NO: 155 (UniProtKB Q13002-5), SEQ ID NO: 156 (UniProtKB Q13002-6), SEQ ID NO: 157 (UniProtKB Q13002-7), SEQ ID NO: 158 (NCBI accession no. : NP_001104738.2), SEQ ID NO: 159 (NCBI accession number: NP_034479.3), SEQ ID NO: 160 (NCBI accession number: NP_034479.3), SEQ ID NO: 161 (NCBI accession number: XP_014992481.1) , the polynucleotide sequences of SEQ ID NO: 162 (NCBI accession number: nucleic acids. The polynucleotide sequence encoding GluK2 receptor can be selected from any of the following: SEQ ID NO: 164-174.

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 151, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 151, which is shown below (UniProt Q13002-1; GRIK2_HUMAN glutamate receptor ionotropic, red Alginate 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAK QTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTEEVIN MHTFNDRRLPKETMA (SEQ ID NO: 151)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 152, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 152, which is shown below (UniProt Q13002-2; glutamate receptor ionotropic species, red algae GRIK2_HUMAN isoform of amino acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAK QTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKESSIWLVPPYHPDTV (SEQ ID NO: 152)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 153, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 153, which is shown below (UniProt Q13002-3; glutamate receptor ionotropic species, red algae GRIK2_HUMAN isoform of amino acid 2 3): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARF (SEQ ID NO: 153)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 154, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 154, which is shown below (UniProt Q13002-4; glutamate receptor ionotropic species, red algae Acid 2 GRIK2_HUMAN isoform 4): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNC NLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTEEVINMHTFNDRRLPGKETMA (SEQ ID NO: 154)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 155, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 155, which is shown below (UniProt Q13002-5; glutamate receptor ionotropic species, red algae GRIK2_HUMAN isoform of amino acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAK Question CS (SEQ ID NO: 155)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 156, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 156, which is shown below (UniProt Q13002-6; glutamate receptor ionotropic species, red algae GRIK2_HUMAN isoform of amino acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNA QLEKESSIWLVPPYHPDTV (SEQ ID NO: 156)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 157, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 157, which is shown below (UniProt Q13002-7; glutamate receptor ionotropic species, red algae GRIK2_HUMAN isoform of amino acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKSVLVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRAKTKLPQDYV FLPILESVSISTVLSSSPSSSSLSSCS (SEQ ID NO: 157)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 158, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 158, which is shown below (NP_001104738.2; glutamate receptor ionotropic, kainate GRIK2_MOUSE of Acid 2 (same type 1 precursor): MKIISPVLSNLVFSRSIKVLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDA KPLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPSSGLNMTESQKGKPANITDSLSNRSLIVTTILEE PYVLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDVNGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTMESPIDSADD LAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTE EVINMHTFNDRRLPGKETMA (SEQ ID NO: 158)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 159, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 159, which is shown below (NP_034479.3; glutamate receptor ionotropic, kainate Acid 2's GRIK2_MOUSE (same type 2 precursor): MKIISPVLSNLVFSRSIKVLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDA KPLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPSSGLNMTESQKGKPANITDSLSNRSLIVTTILEE PYVLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDVNGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTMESPIDSADD LAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKESSIWLVPPYHPDTV (SEQ ID NO: 159)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 160, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 160, which is shown below (NP_001345795.2; glutamate receptor ionotropic, kainate GRIK2_MOUSE of Acid 2 (same type 1 precursor): MKIISPVLSNLVFSRSIKVLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDA KPLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPSSGLNMTESQKGKPANITDSLSNRSLIVTTILEE PYVLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDVNGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTMESPIDSADD LAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTE EVINMHTFNDRRLPGKETMA (SEQ ID NO: 160)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 161, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 161, which is as follows (XP_014992481.1; GRIK2_RHESUS MACAQUE isoform X1, glutamate receptor Ionotropic, kainic acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAK QTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTEEVIN MHTFNDRRLPKETMA (SEQ ID NO: 161)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 162, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 162, which is as follows (XP_014992483.1; GRIK2_RHESUS MACAQUE isoform X1, glutamate receptor Ionotropic, kainic acid 2): MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAK PLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPY VLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAK QTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTEEVIN MHTFNDRRLPKETMA (SEQ ID NO: 162)

The GluK2 polypeptide may have the following amino acid sequence: SEQ ID NO: 163, or may have at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the following amino acid sequence. , 98%, 99% or more (e.g., 100%)) Variants of sequence identity: SEQ ID NO: 163, which is shown below (NP_062182.1; glutamate receptor ionotropic, kainate Acid 2 GRIK2_RAT precursor): MKIISPVLSNLVFSRSIKVLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNTTLTYDTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPDFSSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDA KPLLKEMKRGKEFHVIFDCSHEMAAGILKQALAMGMTEYYHYIFFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAPPKPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPWRFGTRFMSLIKEAHWEGLTGRITFNKTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEE PYVLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDVNGQWNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYVLLACLGVSCVLFVIARFSPYEWYNPHPCNPDSDVVENNFTLLNSFWFGVGALMRQGSELMPKALSTRIVGGIWWFFTLIIISSYTANLAAFLTVERMESPIDSA DDLAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKWWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVK TEEVINMHTFNDRRLPGKETMA (SEQ ID NO: 163)

Grik2 mRNA can be a nucleic acid sequence containing 5' and 3' untranslated regions (UTR) and having SEQ ID NO: 164, or a variant thereof that is at least 85% identical to the following nucleic acid sequence (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) Sequence identity: SEQ ID NO: 164 (RefSeq NM_021956.1:4592 Homo sapiens glutamine Acid ionotropic receptor kainate-type subunit 2 (GRIK2), transcript variant 1, mRNA), as shown in Table 10.

Grik2 mRNA may be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 165, or may be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) Sequence identity: SEQ ID NO: 165 (RefSeq NM_021956.4:294-3020 Homo sapiens glutamate ionotropic receptor red GRIK2 (GRIK2), transcript variant 1, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA can be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 166, or can be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 166 (RefSeq NM_175768.3:294-2903 Homo sapiens glutamate Ionotropic receptor kainate-type subunit 2 (GRIK2), transcript variant 2, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA can be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 167, or can be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 167 (RefSeq NM_001166247.1:294-2972 Homo sapiens glutamate Ionotropic receptor kainate-type subunit 2 (GRIK2), transcript variant 3, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA may be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 168, or may be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 168 (RefSeq NM_001111268.2 House Mole ( Mus musculus ) Glutamine Acid ionotropic receptor kainate subunit 2 (GRIK2), transcript variant 4, mRNA), is shown below.

Additionally or alternatively, Grik2 mRNA may be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 169, or may be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 169 (RefSeq NM_010349.4 House Mole Glutamate Ionotropic Receptor kainate subunit 2 (GRIK2), transcript variant 5, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA may be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 170, or may be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 170 (RefSeq NM_ 001358866 House Mole Glutamate Ionotropic Receptor body kainate subunit 2 (GRIK2), transcript variant 6, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA can be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 171, or can be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 171 (RefSeq XM_015136995.2 Rhesus macaque ( Macaca mulatta ) Glutamine Acid ionotropic receptor kainate subunit 2 (GRIK2), transcript variant 7, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA may be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 172, or may be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) Sequence identity: SEQ ID NO: 172 (RefSeq XM_015136997.2 Rhesus macaque glutamate ionotropic Receptor kainate subunit 2 (GRIK2), transcript variant X1, mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA can be a polynucleotide having the nucleic acid sequence of SEQ ID NO: 173, or can be a variant thereof that is at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 173 (RefSeq NM_019309.2 Rattus norvegicus glutamate-stimulating Ionotropic receptor kainate subunit 2 (GRIK2), mRNA), as shown in Table 10.

Additionally or alternatively, Grik2 mRNA includes a polynucleotide corresponding to the mature GluK2 peptide coding sequence and having the nucleic acid sequence of SEQ ID NO: 174 or a variant thereof that shares at least 85% of the nucleic acid sequence of SEQ ID NO: 174 % (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity, as shown in Table 10.

According to the disclosed methods and compositions, Grik2 mRNA may include a 5' UTR, such as a 5' UTR encoded by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 175 or a variant thereof that is identical to SEQ ID NO: 175 nucleic acid sequences having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity, as shown in Table 10 shown.

Grik2 mRNA may also include a 3' UTR, such as a 3' UTR encoded by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 176 or a variant thereof that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 176 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity, as shown in Table 10.

In addition, Grik2 mRNA may include a polynucleotide encoding a Grik2 message peptide sequence, for example, a message peptide sequence encoded by a polynucleotide of the nucleic acid sequence of SEQ ID NO: 177 or a variant thereof, which variant is identical to SEQ ID NO: 177 The nucleic acid sequence has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity, as set forth in Table 10 Show.

In addition, the inhibitory polynucleotide of the present disclosure can bind within any of exons 1 to 16 (corresponding to SEQ ID NO: 177 to SEQ ID NO: 193) of the Grik2 transcript, which exons Described in Table 10 below. Table 10: cDNA sequences encoding target Grik2 mRNA sequences instruction Biological source RefSeq SEQ ID NO Grik2 mRNA, variant 1; includes 5' UTR and 3' UTR Homo sapiens NM_021956.1:4592 164 Grik2 mRNA, variant 1; (CDS only) Corresponds to nt positions 294 to 3020 of SEQ ID NO: 116. Homo sapiens NM_021956.294-3020 165 Grik2 mRNA, variant 2 Homo sapiens NM_175768.3:294-2903 166 Grik2 mRNA, variant 3 Homo sapiens NM_001166247.1:294-2972 167 Grik2 mRNA, variant 4 Mus musculus NM_001111268 168 Grik2 mRNA, variant 5 Mus musculus NM_010349 169 Grik2 mRNA, variant 6 Mus musculus NM_001358866 170 Grik2 mRNA, variant 7 rhesus macaque XM_015136995.2 171 Grik2 mRNA, variant 8 rhesus macaque XM_015136997.2 172 Grik2 mRNA, variant 9 ditch rat NM_019309.2 173 Grik2 mRNA encoding mature GluK2 peptide CDS Homo sapiens NM_021956.1:4592 174 Grik2 mRNA, 5' UTR Homo sapiens NM_021956.1:4592 175 Grik2 mRNA, 3' UTR Homo sapiens NM_021956.1:4592 176 Grik2 mRNA, message peptide sequence Homo sapiens NM_021956.1:4592 177 Grik2 mRNA, exon 1; SEQ ID NO: nt 1 to 408 of 151 Homo sapiens NM_021956.1:4592 178 Grik2 mRNA, exon 2; SEQ ID NO: nt 409 to 576 of 151 Homo sapiens NM_021956.1:4592 179 Grik2 mRNA, exon 3; nt 577 to 834 of SEQ ID NO: 151 Homo sapiens NM_021956.1:4592 180 Grik2 mRNA, exon 4; SEQ ID NO: 151-nt 835 to 1016 Homo sapiens NM_021956.1:4592 181 Grik2 mRNA, exon 5; SEQ ID NO: 151 to nt 1017 to 1070 Homo sapiens NM_021956.1:4592 182 Grik2 mRNA, exon 6; SEQ ID NO: 151 to nt 1071 to 1244 Homo sapiens NM_021956.1:4592 183 Grik2 mRNA, exon 7; SEQ ID NO: 151 to nt 1245 to 1388 Homo sapiens NM_021956.1:4592 184 Grik2 mRNA, exon 8; SEQ ID NO: 151-nt 1389 to 1496 Homo sapiens NM_021956.1:4592 185 Grik2 mRNA, exon 9; SEQ ID NO: 151-nt 1497 to 1610 Homo sapiens NM_021956.1:4592 186 Grik2 mRNA, exon 10; SEQ ID NO: 151-nt 1611 to 1817 Homo sapiens NM_021956.1:4592 187 Grik2 mRNA, exon 11; SEQ ID NO: 151-nt 1818 to 2041 Homo sapiens NM_021956.1:4592 188 Grik2 mRNA, exon 12; SEQ ID NO: 151-nt 2042 to 2160 Homo sapiens NM_021956.1:4592 189 Grik2 mRNA, exon 13; SEQ ID NO: 151-nt 2161 to 2378 Homo sapiens NM_021956.1:4592 190 Grik2 mRNA, exon 14; SEQ ID NO: 151-nt 2379 to 2604 Homo sapiens NM_021956.1:4592 191 Grik2 mRNA, exon 15; SEQ ID NO: 151-nt 2605 to 2855 Homo sapiens NM_021956.1:4592 192 Grik2 mRNA, exon 16; SEQ ID NO: 151-nt 2856 to 4592 Homo sapiens NM_021956.1:4592 193 Nucleic acid vector

Effective intracellular concentrations of the nucleic acid agents disclosed herein can be achieved through stable expression of the polynucleotide encoding the agent (eg, by integration into the nuclear or mitochondrial genome of a mammalian cell). The nucleic acid is an inhibitory RNA that targets Grik2 mRNA (eg, an inhibitory RNA agent disclosed herein). To introduce such exogenous nucleic acids into mammalian cells, the polynucleotide sequence of the agent can be incorporated into a vector. Vectors can be introduced into cells by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described in more detail in, for example, Green et al., Molecular Cloning: A Laboratory Manual, 4th ed. (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)), whose respective disclosures are incorporated herein by reference.

The agents disclosed herein can also be introduced into mammalian cells by targeting a vector containing a polynucleotide encoding such agent to cell membrane phospholipids. For example, the vector can be targeted to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Accordingly, constructs may be generated using methods customary and routine in the art.

In addition to achieving high transcription and translation rates, the stable expression of exogenous polynucleotides in mammalian cells can be achieved by integrating the polynucleotide containing the gene into the nuclear genome of the mammalian cells. A variety of vectors have been developed for the delivery and integration of polynucleotides encoding foreign proteins into the nuclear DNA of mammalian cells. Examples of expression vehicles are disclosed, for example, in WO 1994/011026 and incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain polynucleotide sequences encoding Grik2- targeting inhibitory RNA agents and, for example, are used to express such agents and/or integrate such polynucleotide sequences into mammals. Additional sequence elements in the genome of a cell. Some vectors that can be used include plasmids containing regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other useful vectors contain polynucleotide sequences that increase the translation rate of such genes or enhance the stability or nuclear export of the mRNA responsible for gene transcription. Such sequence elements include, for example, 5' and 3' UTR regions, IRES, and polyadenylation message sequence sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, conmycin, nourseothricin. regulatory sequence

The inhibitory RNA agents disclosed herein can perform at sufficiently high levels to elicit therapeutic benefit. Accordingly, polynucleotide expression may be mediated by promoter sequences capable of driving robust expression of the disclosed inhibitory RNA agents. According to the methods and compositions disclosed herein, the promoter can be a heterologous promoter. As used herein, the term "heterologous promoter" refers to a promoter not found in nature operably linked to a given coding sequence. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.

Both heterologous promoters and other control elements can be used, such as CNS-specific and inducible promoters, enhancers, and the like. The promoter may be derived entirely from a native gene (eg, the Grik2 gene) or may be composed of different elements derived from different naturally occurring promoters. Alternatively, the promoter may comprise a synthetic polynucleotide sequence. Different promoters will direct the expression of genes in different tissues or cell types, or at different stages of development, or in response to different environmental conditions, or in response to the presence or absence of drugs or transcription cofactors. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific and conditional promoters, eg, drug-responsive promoters (eg, tetracycline-responsive promoters), are well known in the art.

In mammalian systems, three promoters exist and are candidate promoters for constructing expression vectors: (i) the Pol I promoter that controls the transcription of large ribosomal RNA; (ii) the Pol I promoter that controls mRNA (translation into protein), small Pol II promoters that transcribe nuclear RNAs (snRNAs) and endogenous microRNAs (eg, from introns in pre-mRNAs); (iii) and Pol III promoters that uniquely transcribe small non-coding RNAs. Each of these promoters has advantages and limitations that need to be considered when designing constructs for in vivo RNA expression. For example, the Pol III promoter can be used to synthesize inhibitory RNA agents (e.g., siRNA, shRNA, miRNA, or shmiRNA) from DNA templates in vivo. For better control of tissue-specific expression, Pol II promoters can be used (e.g., for miRNA transcription). When using the Pol II promoter, the translation initiation message can be omitted, allowing the RNA to function as siRNA, shRNA, or miRNA and not be translated into peptides in vivo.

Polynucleotides suitable for use in the compositions and methods described herein also include those encoding inhibitory RNA agents found in mammalian regulatory sequences, such as promoter sequences and, optionally, enhancer sequences. Targets Grik2 mRNA under the control of Exemplary promoters useful for expression of the disclosed inhibitory RNA agents in mammalian cells include cell type-specific promoters. For example, neuron-specific expression of Grik2 inhibitory RNA agents can be conferred using neuron-specific promoters, such as the human synapsin 1 (hSyn) promoter or the Ca ²⁺ /calcin-dependent protein kinase II (CaMKII ) promoter. Variants of the hSyn and CaMKII promoters have been previously described in Hioki et al. Gene Therapy 14:872-82 (2007) and Sauerwald et al. J. Biol. Chem. 265(25):14932-7 (1990) , the disclosures of which are incorporated herein by reference as they relate to specific hSyn and CaMKII promoter sequences. Constitutive promoters containing the cytomegalovirus enhancer (eg, CAG or CBA), U6, H1, or 7SK promoters can also be used. The sequences of such promoters are known in the art (the sequences of such promoters are also disclosed, for example, in WO 2022/011262, which is incorporated herein by reference).

In specific examples, expression vectors of the present disclosure include a SYN promoter (such as the human SYN promoter (hSyn), for example, any of the following: SEQ ID NO: 194 to SEQ ID NO: 198 or a variant thereof, which variant 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 with any of the following Identity: SEQ ID NO: 194-198). In another example, expression vectors of the present disclosure include a CAMKII promoter (e.g., any of the following: SEQ ID NO: 199 to SEQ ID NO: 204 or a variant thereof that is consistent with any of the following The nucleic acid sequence has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity: SEQ ID NO: 99-204).

Exemplary promoter sequences suitable for expression vectors (eg, plasmid or viral vectors, such as AAV or lentiviral vectors) are provided in Table 11 below. Table 11 : Exemplary neuron-specific promoter sequences promoter SEQ ID NO Nucleotide sequence GenBank RefSeq Syn (short_1; Homo sapiens ) 194 ctgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcactgccagcttcagcaccgcggac agtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggcacgggcgcgaccatctg cgctgcggcgccggcgactcagcgctgcctcagtctgc M55301.1 Syn (short_2; Homo sapiens ) 195 ctgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcactgccagcttcagcaccgcggac agtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggcacgggcgcgaccatctg cgctgcggcgccggcgactcagcgctgcctcagtctgccaattgcagcggaggagtcgtgtcgtgcctgagagcgcag M55301.1 Syn (short_2.5; Homo sapiens ) 196 CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCCGCTGA CGTCACTCGCCGGTCCCCCGCAAACTCCCCTCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCCAATTGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGGGCGCGCC M55301.1 Syn (short_3; Homo sapiens ) 197 CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCCGCTGA CGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCG M55301.1 Syn (long_1; Homo sapiens ) 198 ctgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcactgccagcttcagcaccgcggac agtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggcacgggcgcgaccatctg cgctgcggcgccggcgactcagcgctgcctcagtctgcGGTGGgcagcggaggagtcgtgtcgtgcctgagagcgcagggcgcgcc M55301.1 CaMKII_1 199 CATTATGGCCTTAGGTCACTTCATCTCCATGGGGTTCTTCTCTGATTTTCTAGAAAATGAGATGGGGGTGCAGAGAGCTTCCTCAGTGACCTGCCCAGGGTCACATCAGAAATGTCAGAGCTAGAACTTGAACTCAGATTACTAATCTTAAATTCCATGCCTTGGGGGCATGCAAGTACGATATACAGAAGGAGTGAACTCATTAGGGCAGATGACCAATGAGTTTAGGAAAGAAGAGTCCAGGGCAGGGTACATCTAC ACCACCCGCCCAGCCCTGGGTGAGTCCAGCCACGTTCACCTCATTATAGTTGCCTCTCCAGTCCTACCTTGACGGGAAGCACAAGCAGAAACTGGGACAGGAGCCCCAGGAGACCAAATCTTCATGGTCCCTCTGGGAGGATGGGTGGGGAGCTGTGGCAGAGGCCTCAGGAGGGGCCCTGCTGCTCAGTGGTGACAGATAGGGGTGAGAAAGCAGACAGAGTCATTCCGTCAGCATTCTGGGTCTGTTTGGTACTTCTTCT CACGCTAAGGTGGCGGTGTGATATGCACAATGGCTAAAAAGCAGGGAGCTGGAAAGAAACAAGGACAGAGACAGAGGCCAAGTCAACCAGACCAATTCCCAGAGGAAGCAAAGAAACCATTACAGAGACTACAAGGGGGAAGGGAAGGAGAATGAATTAGCTTCCCCTGTAAACCTTAGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTCTTCAGAGGAGATGAAGTTGCCAGGGTAACTACATCCTGTCTTTCT CAAGGACCATCCCAGAATGTGGCACCCACTAGCCGTTACCATAGCAACTGCCTCTTTGCCCCACTTAATCCCATCCCGTCTGTTAAAAGGGCCCTATAGTTGGAGGTGGGAGGTAGGAAGAGCGATGATCACTTGTGGACTAAGTTTGTTCGCATCCCCTTCTCCAACCCCCTCAGTACATCACCCTGGGGGAACAGGGTCCACTTGCTCCTGGGCCCACACAGTCCTGCAGTATTGTGTATATAAGGCCAGGGCAAAGAGGAGCAGGTT TTAAAGTGAAAGGCAGGCAGGTGTTGGGGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTCGGAGGTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGCCACAGTGCCCTGCTCAGAAGCCCCAAGCTCGTCAGTCAAGCCGGTTCTCCGTTTGCACTCAGGAGCACGGGCAGGCGAGTGGCCCCTAGTTCTGGGGGCAGC AJ222796.1 CaMKII (α; Homo sapiens ) 200 gatgctgacgaaggctcgcgaggctgtgagcagccacagtgccctgctcAGAAGCCCCGG NM_171825 CaMKII (β1; Homo sapiens ) 201 gtctcccgcgcccgcgcccgtgtcgccgccgtgcccgcgagcgggagccGGAGTCGCCGC NM_172084 CaMKII (β2; Homo sapiens ) 202 cgtgtgcagatgcagggcgccggtgccctgcgggtgcgggtgcaggagcAGCGTGGTGCAG NM_172084 CaMKII (delta; Homo sapiens ) 203 ccccacgccaccctttctggtcatctcccctcccgccccgcccctgcgcACACTCCCTCG NM_172115 CaMKII (γ; Homo sapiens ) 204 tctccccggtaaagtctcgcggtgctgccgggctcagccccgtctcctcCTCTTGCTCCC NM_172171

In certain examples, viral vectors of the present disclosure incorporate neuron-specific promoter sequences. In a specific example, the neuron-specific promoter is a human Syn (hSyn) promoter, such as a human Syn promoter having a nucleic acid sequence of any of: SEQ ID NO: 194 to SEQ ID NO: 198, or thereof A variant that shares at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%) the nucleic acid sequence of any of the following , 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 194-198.

In another example, the neuron-specific promoter is a CaMKII promoter sequence, such as a CaMKII promoter sequence of any of: SEQ ID NO: 199 to SEQ ID NO: 204, or a variant thereof, which variant A nucleic acid sequence that is at least 70% identical to any of the following (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 199-204.

Additional CaMKII promoters may include the human alpha CaMKII promoter sequence described in Wang et al. ( Mol. Biol. Rep. 35(1):37-44, 2007), the disclosure of which is incorporated herein in its entirety. Because it involves the CaMKII promoter sequence.

Once a polynucleotide encoding a disclosed inhibitory RNA agent is incorporated into the nuclear DNA of a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical agents, such as agents that modulate the binding of transcription factors and/or RNA polymerases to mammalian promoters and thereby modulate gene expression. Chemical reagents can be used to promote binding of RNA polymerase and/or transcription factors to mammalian promoters, for example, by removing repressor proteins that have bound to the promoter. Alternatively, chemical reagents can be used to enhance the affinity of a mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of genes located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical agents that enhance polynucleotide transcription through the above mechanisms are tetracycline and deoxytetracycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

Other DNA sequence elements that may be included in the polynucleotides used in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in the polynucleotide containing the target gene such that the DNA adopts a three-dimensional orientation that favors the binding of transcription factors to RNA polymerase at the transcription start site. Accordingly, polynucleotides for use in the compositions and methods described herein include those encoding Grik2- targeting inhibitory RNA agents and additionally include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, and examples are enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples are the SV40 enhancer (bp 100-270) located behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma virus enhancer located behind the origin of replication, and the adenovirus enhancer. Additional enhancer sequences that induce activation of eukaryotic gene transcription were revealed in Yaniv et al., Nature 297:17 (1982). The enhancer can be spliced into a vector containing a polynucleotide encoding an antisense construct of the present disclosure, for example, at a position 5' or 3' relative to the gene. In a particular orientation, the enhancer is located 5' to the promoter, which in turn is located 5' to the polynucleotide encoding the inhibitory RNA agent of the present disclosure. Non-limiting examples of enhancer sequences are provided in Table 12.

Other regulatory elements that may be included in the polynucleotides used in the compositions and methods described herein are intronic sequences. Intronic sequences are non-protein-coding RNA sequences found in pre-mRNA that are removed during RNA splicing to produce the mature mRNA product. Intronic sequences are important in the regulation of gene expression because they can be further processed to produce other noncoding RNA molecules. Alternative splicing, nonsense-mediated decay, and mRNA export are biological processes that have been shown to be regulated by intronic sequences. Intronic sequences may also promote transgenic gene expression through intron-mediated enhancement. Non-limiting examples of intron sequences are provided in Table 12.

Other regulatory elements that may be used in conjunction with the vectors of the present disclosure include inverted terminal repeat (ITR) sequences. ITR sequences are found, for example, at the 5' and 3' ends of the AAV genome, and these sequences typically contain approximately 145 base pairs each. The AAV ITR sequence is particularly important for AAV genome multiplication by promoting complementary strand synthesis once the AAV vector is incorporated into the cell. Furthermore, ITRs have been shown to be critical for integration of AAV gene bodies into host cell gene bodies and for encapsidation of AAV gene bodies. Non-limiting examples of ITR sequences are provided in Table 12.

Other regulatory elements suitable for incorporation into vectors of the present disclosure include polyadenylation sequences (ie, polyA sequences). The PolyA sequence is an RNA tail containing a stretch of adenine bases. These sequences are appended to the 3' end of the RNA molecule to produce the mature mRNA transcript. Several biological processes related to mRNA processing and transport, including nuclear export, translation, and stability, are regulated by polyA sequences. In mammalian cells, shortening the polyA tail leads to an increased likelihood of mRNA degradation. Non-limiting examples of polyA sequences are provided in Table 12 below. Table 12 : Exemplary regulatory sequences Adjustment element SEQ ID NO Nucleotide sequence chimeric intron 205 GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG VH4 intron 206 GTGAGTATCTCAGGGATCCAGACATGGGGATATGGGAGGTGCCTCTGATCCCAGGGCTCACTGTGGGTCTCTCTGTTCACAG CMV enhancer 207 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTG GCAGTACATCTACGTATTAGTCATCGCTATTACCATG AAV 5'-ITR 208 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Modified AAV 5'-ITR (D sequence deletion for self-complementary AAV) 209 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG AAV 3'-ITR 1 210 GAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA AAV 3'-ITR 2 212 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG Modified AAV 3'-ITR (D sequence deletion for self-complementary AAV) 211 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG Rabbit Beta-Globin (RBG) Poly(A) Message #1 213 ATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA RBG PolyA #2 214 GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG RBG PolyA #3 215 GATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG Bovine Growth Hormone (BGH) PolyA 216 TAGCAGGCATGCTGGGGAG

In other examples, the viral vectors of the present disclosure incorporate one or more regulatory sequence elements capable of promoting the expression of the antisense constructs of the present disclosure. In one example, the regulatory sequence elements are intronic sequences. For example, an intron sequence suitable for inclusion in a vector of the present disclosure may be a chimeric intron, such as a chimeric intron having the following nucleic acid sequence: SEQ ID NO: 205 or a variant thereof, which variant Be at least 70% identical to the nucleic acid sequence of SEQ ID NO: 205 (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity.

In another example, the intron sequence is an immunoglobulin heavy chain variable 4 (VH4) intron, such as a VH4 sequence having the following nucleic acid sequence: SEQ ID NO: 206 or a variant thereof that is identical to The nucleic acid sequence of SEQ ID NO: 206 has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more (e.g., 100%)) sequence identity.

In some embodiments, from 5' to 3', the vector includes: (a) a first promoter sequence; (b) an intron sequence; (c) a polynucleotide comprising a stem-loop sequence; (d) optionally , a second promoter sequence; and (e) optionally, a polynucleotide comprising a stem-loop sequence.

In another example, the regulatory sequence element is an enhancer sequence. For example, the enhancer sequence may be a CMV enhancer, such as a CMV enhancer having the nucleic acid sequence of SEQ ID NO: 207 or a variant thereof that is at least 70% identical to the nucleic acid sequence of SEQ ID NO: 207 (e.g., , at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100 %)) sequence identity.

In some embodiments, from 5' to 3', the vector includes: (a) an enhancer sequence; (b) a first promoter sequence; (c) an intron sequence; (d) a polynucleoside containing a stem-loop sequence acid; (e) optionally, a second promoter sequence; and (f) optionally, a polynucleotide comprising a stem-loop sequence.

In another example, the regulatory sequence element is an ITR sequence, such as an AAV ITR sequence. For example, the ITR sequence may be an AAV 5' ITR sequence, such as an AAV 5' ITR sequence having the following nucleic acid sequence: SEQ ID NO: 208 or SEQ ID NO: 209 or a variant thereof that is identical to the following nucleic acid sequence At least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity: SEQ ID NO: 208 or SEQ ID NO: 209.

In another example, the ITR sequence is an AAV 3' ITR sequence, such as an AAV 3' ITR sequence having the nucleic acid sequence of any of: SEQ ID NO: 210 to SEQ ID NO: 212, or a variant thereof, The variant has a nucleic acid sequence that is at least 70% identical to any of the following (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 210-212.

In some embodiments, the vector includes: from 5' to 3', (a) a 5' ITR sequence; (b) optionally, an enhancer sequence; (c) a first promoter sequence; (d) optionally, within intron sequence; (e) a polynucleotide containing a stem-loop sequence; (f) optionally a second promoter sequence; (g) optionally a polynucleotide containing a stem-loop sequence; and (h) 3' ITR .

In another example, the regulatory sequence element is a polyadenylation message sequence (ie, a polyadenylation tail). For example, polyadenylation messages suitable for use with the vectors disclosed herein include rabbit beta-globin (RBG) polyadenylation messages, such as RBG polyadenylation having a nucleic acid sequence of any of the following Message sequence: SEQ ID NO: 213 to SEQ ID NO: 215 or a variant thereof, which is at least 70% (for example, at least 70%, 75%, 80%, 85%) identical to any of the following nucleic acid sequences %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence Identity: SEQ ID NO: 213 -215. Another polyadenylation message that may be used in conjunction with the disclosed compositions and methods is the bovine growth hormone (BGH) polyadenylation message, such as the following BGH polyadenylation message: SEQ ID NO: 216 Or a variant thereof, which has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%) the nucleic acid sequence of SEQ ID NO: 216 %, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity. In some embodiments, from 5' to 3', the vector includes: (a) 5' ITR sequence; (b) optionally, enhancer sequence; (c) first promoter sequence; (d) optionally, within Intron sequence; (e) Polynucleotide containing a stem-loop sequence; (f) Optionally, a second promoter sequence; (g) Optionally, a polynucleotide containing a stem-loop sequence; (h) Polyadenylation message sequence, such as the RBG polyadenylation message sequence; and (i) 3' ITR. viral vector

Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous polynucleotides into mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the nuclear genome of mammalian cells by general or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors are parvovirus (eg, adeno-associated virus (AAV)), retrovirus (eg, Retroviridae vectors), adenovirus (eg, Ad5, Ad26, Ad34, Ad35 and Ad48), coronavirus , negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-stranded RNA viruses (e.g., measles virus and Sendai virus), such as picornaviruses and alphaviruses) and double-stranded DNA viruses (including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Estén-Barr virus, cytomegalovirus) and pox Viruses (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), fowlpox virus, and canarypox virus)). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papillomavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus. Examples of retroviruses are avian leukemic sarcoid virus, avian C virus, mammalian C virus, B virus, D virus, oncogenic retrovirus, HTLV-BLV group, lentivirus, alpha retrovirus, gamma retrovirus Retrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, 3rd ed. (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia virus, murine sarcoma virus, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus , Mason Pfizer simian virus, simian immunodeficiency virus, simian sarcoma virus, Rous' sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in McVey et al. (U.S. Patent No. 5,801,030), the teachings of which are incorporated herein by reference.

AAV carrier

The nucleic acids of the compositions described herein can be incorporated into AAV vectors and/or viral particles to facilitate their introduction into cells, for example, in conjunction with the methods disclosed herein. AAV vectors can be used in the central nervous system, and appropriate promoters and serotypes are discussed, for example, in Pignataro et al., J Neural Transm 125(3):575-89 (2017), the disclosure of which is incorporated herein by reference as it Involves promoters and AAV serotypes useful in CNS gene therapy. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a heterologous sequence to be expressed (e.g., a polynucleotide encoding a Grik2 mRNA-targeting inhibitory RNA agent) and ( 2) Viral sequences that promote integration and expression of heterologous genes. Such viral sequences may include sequences of their AAVs required for cis replication and packaging of DNA into virions (eg, functional ITRs). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITR can be any serotype suitable for a specific application. Methods using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), as they relate to AAV vectors for gene delivery. Their respective disclosures are incorporated herein by reference.

Useful for incorporation into nucleic acid agents described herein (e.g., inhibitory RNA sequences (e.g., SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to any one of SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229 and SEQ ID NO: 238 to SEQ ID NO: 241)) Examples of AAV vectors include, for example, 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-TT, AAV-DJ8 or AAV.HSC16.

The nucleic acids and vectors described herein can be incorporated into rAAV viral particles to facilitate the introduction of the nucleic acids or vectors into cells. The capsid protein of AAV constitutes the outer non-nucleic acid part of the virus particle and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virus particle assembly. The construction of rAAV virions has been described, for example, in US 5,173,414, US 5,139,941, US 5,863,541, US 5,869,305, US 6,057,152 and US 6,376,237, and Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al. , J. Virol. 77:423 (2003), the respective disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

rAAV virions for use in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and rh74. virus particles. AAV2, AAV9, and AAV10 may be particularly useful for targeting cells located in or delivered to the central nervous system. The construction and use of AAV vectors and AAV proteins of different serotypes are described in, for example, Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci.USA 97: 3428 (2000); Xiao et al., J. Virol.72:2224 (1998); Halbert et al., J. Virol.74:1524 (2000); Halbert et al., J. Virol.75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

Pseudotyped rAAV vectors may also be used in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype pseudotyped with capsid genes derived from serotypes other than the given serotype (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-TT, AAV-DJ8 or AAV .HSC16). For example, AAV may include pseudotyped recombinant AAV (rAAV) vectors, such as rAAV2/8 or rAAV2/9 vectors. Methods for generating and using pseudotyped rAAV are known in the art (see, eg, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74 :1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

AAV virions with mutations within the virion capsid can be used to infect specific cell types more efficiently than unmutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to facilitate targeting of AAV to specific cell types. Construction and characterization of AAV capsid mutants, including insertion mutants, alanine selection mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions useful in the methods described herein include those capsid hybrids produced by molecular breeding of the virus and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

rAAV used in the compositions and methods of the present disclosure may include capsid proteins from AAV capsid serotypes selected from: 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-TT, AAVDJ8 or its derivatives , modified or pseudotyped, such as at least 80% or more identical to the sequence of vp1, vp2 and/or vp3 of an AAV capsid serotype selected from the following (e.g., 85%, 85%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, that is, up to 100% identical) of the shell protein : 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-TT, AAV-DJ8 or AAV.HSC16.

AAV vectors useful in the methods described herein may be Anc80 or Anc80L65 vectors, as described in Zinn et al., 2015: 1056-1068, which is incorporated by reference in its entirety. AAV vectors may include one of the following amino acid insertions: LGETTRP (SEQ ID NO: 14 of '956, '517, '282, or '323) or LALGETTRP (SEQ of '956, '517, '282, or '323 ID NO: 15), as described in U.S. Patent Nos. 9,193,956, 9458517, and 9,587,282, and U.S. Patent Application Publication No. 2016/0376323, each of which is incorporated herein by reference in its entirety. Alternatively, the AAV vector used in the methods described herein can be AAV.7m8, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282 and U.S. Patent Application Publication No. 2016/0376323, each of which is incorporated by reference in its entirety. Incorporated herein. Still further, the AAV vector used in the methods described herein can be any AAV disclosed in US Pat. No. 9,585,971, such as the AAV.PHP.B vector. Another AAV vector used in the methods described herein can be any of the vectors disclosed in Chan et al. ( Nat Neurosci. 20(8):1172-1179, 2017), such as AAV.PHP.eB, which contains an amine Peptides between amino acid positions 588 and 589 and modified A587D/588G AAV9 capsid protein. Additionally, the AAV vector used in the methods described herein can be any AAV disclosed in U.S. Patent No. 9,840,719 and WO 2015/013313, such as the AAV.Rh74 or RHM4-1 vector, each of which is incorporated by reference in its entirety. This article. Furthermore, the AAV vector used in the methods described herein can be any AAV disclosed in WO 2014/172669, such as AAV rh.74, which patent is incorporated herein by reference in its entirety. The AAV vector used in the methods described herein can also be an AAV2/5 vector, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450; these documents Each is incorporated by reference in its entirety. In yet another example, the AAV vector used in the methods described herein can be any AAV disclosed in WO 2017/070491, such as the AAV2tYF vector, which patent is incorporated herein by reference in its entirety. Additionally, the AAV vector used in the methods described herein can be an AAVLK03 or AAV3B vector, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. . In other examples, the AAV vector used in the methods described herein can be any AAV disclosed in US Pat. No. 8,628,966, US 8,927,514, US 9,923,120, and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15 or HSC16, each of these patents is incorporated by reference in its entirety.

In addition, the AAV vector used in the methods described herein can be the AAV vector disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517 and 9,587,282; US Patent Application Publication No. 2015/0374803, 2015 /0126588, 2017/0067908, 2013/0224836, 2016/0215024, 2017/0051257; and international patent application numbers PCT/US2015/034799, PCT/EP2015/053335. The rAAV vector may have a capsid protein that is consistent with the vp1, vp2, and/or vp3 amino acid sequences of the AAV capsid disclosed in the following patents and patent applications (each of which is incorporated herein by reference in its entirety) At least 80% or more the same (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, 99.5% or more (e.g., 100%)): U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US 9,284,357, 9,409,953, 9,16 9,299, 9,193,956, 9458517 and 9,587,282; U.S. Patents Application publication numbers 2015/0374803, 2015/0126588, 2017/0067908, 2013/0224836, 2016/0215024, 2017/0051257; and international patent application numbers PCT/US2015/034799, PCT/EP2015/053335.

In addition, the rAAV vector may have a capsid protein disclosed in the following documents: International Application Publication No. WO 2003/052051 (see, for example, SEQ ID NO: 2 of '051), WO 2005/033321 (see, for example, '321 of SEQ ID NO: 123 and SEQ ID NO: 88), WO 03/042397 (see, for example, SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 85 and SEQ ID NO: 97 of '397), WO 2006/068888 (see, for example, SEQ ID NO: 1 and SEQ ID NO: 3 to SEQ ID NO: 6 of '888), WO 2006/110689 (see, for example, SEQ ID NO: 5 to SEQ ID NO: 689 of '689) ID NO: 38), WO2009/104964 (see, for example, SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO of '964 : 22, SEQ ID NO: 24 and SEQ ID NO: 31), WO 2010/127097 (see, for example, SEQ ID NO: 5 to SEQ ID NO: 38 of '097) and WO 2015/191508 (see, for example, SEQ ID NO: 5 to SEQ ID NO: 38 of '097) SEQ ID NO: 80 to SEQ ID NO: 294 of '058) and U.S. Application Publication No. 20150023924 (see, e.g., SEQ ID NO: 1, SEQ ID NO: 5 to SEQ ID NO: 10 of '924), each of which The contents of are incorporated herein by reference in their entirety. For example, the rAAV vector has at least 80% (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more (for example, 100%) ) Identical capsid protein: International Application Publication No. WO 2003/052051 (see, for example, SEQ ID NO: 2 of '051), WO 2005/033321 (see, for example, SEQ ID NO: 123 and SEQ ID NO. of '321 NO: 88), WO 03/042397 (see, e.g., SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 85 and SEQ ID NO: 97 of '397), WO 2006/068888 (see, e.g., SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 85 and SEQ ID NO: 97 of '397) , SEQ ID NO: 1 and SEQ ID NO: 3 to SEQ ID NO: 6 of '888), WO 2006/110689 (see, for example, SEQ ID NO: 5 to SEQ ID NO: 38 of '689), WO2009/ 104964 (See, e.g., SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 of '964 and SEQ ID NO: 31), WO 2010/127097 (see, for example, SEQ ID NO: 5 to SEQ ID NO: 38 of '097) and WO 2015/191508 (see, for example, SEQ ID NO: 80 of '508 to SEQ ID NO: 294) and U.S. Application Publication No. 20150023924 (see, e.g., SEQ ID NO: 1, SEQ ID NO: 5 to SEQ ID NO: 10 of '924).

The nucleic acid sequences of AAV-based viral vectors and methods for preparing recombinant AAV and AAV capsids are as taught in the following documents: US Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US 9,284,357, 9,409,9 53, 9,169,299, 9,193,956, 9458517 and 9,587,282; U.S. Patent Application Publication Nos. 2015/0374803, 2015/0126588, 2017/0067908, 2013/0224836, 2016/0215024, 2017/0051257; International Patent Application Nos. PCT/US2015/034799, PC T/EP2015/053335、WO 2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689, WO2009/104964, WO 2010/127097 and WO 2015/191508; and U.S. Application Publication No. 201500239 No. 24.

Accordingly, the rAAV vector may comprise a capsid containing capsid proteins from two or more AAV capsid serotypes, such as an AAV serotype selected from: 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- TT, AAV-DJ8 or AAV.HSC16.

Single-stranded AAV (ssAAV) vectors can be used in conjunction with the disclosed methods and compositions. Alternatively, self-complementary AAV vectors (scAAV) can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al. 2001, Gene Therapy, Vol. 8, No. 16, pages 1248-1254; and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

Recombinant AAV vectors with tropism for cells in the central nervous system, including but not limited to neurons and/or glial cells, can be used to deliver polynucleotide agents (eg, inhibitory RNA agents) of the present disclosure. Such vectors may include non-replicating "rAAV" vectors, specifically vectors with AAV5, AAV9 or AAVrh10 capsids. AAV variant shells may be used, including, but not limited to, those described by Wilson in U.S. Patent No. 7,906,111, which is incorporated by reference in its entirety, designated AAV/hu.31 and AAV/hu. 32 is particularly preferred; and the AAV variant shells described by Chatterjee in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, and Smith et al., 2014, Mol Ther 22: 1625-1634, each of which is Incorporated by reference in its entirety. In addition, the AAV-TT vector disclosed by Tordo et al. ( Brain 141:2014-31, 2018; incorporated herein by reference in its entirety) incorporates conserved amino acid sequences in natural AAV2 isolates and can also Use in conjunction with the compositions and methods of the present disclosure. Compared to AAV2, AAV9, and AAVrh10, AAV-TT variant capsids exhibit improved neurotropism and robust distribution throughout the CNS. Similarly, the AAV-DJ8 vector disclosed by Hammond et al. ( PLoS ONE 12(2):e0188830, 2017; incorporated herein by reference in its entirety) exhibits excellent neurotropism and may be suitable for compositions with the present disclosure. and methods are applicable.

In specific examples, the present disclosure features AAV9 vectors, including artificial genomes, including (i) containing inhibitory RNA sequences (e.g., SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229 and SEQ ID NO: 238 to SEQ ID NO : any of 241) a polynucleotide expression cassette that is under the control of a regulatory element and flanked by an ITR; and (ii) a viral capsid that has the amino acid sequence of the AAV9 capsid protein or is identical to The amino acid sequence of the AAV9 capsid protein is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical, while retaining the biological function of the AAV9 capsid. The encoded AAV9 capsid may have the sequence of SEQ ID NO: 116 set forth in U.S. Patent No. 7,906,111, which is incorporated by reference in its entirety, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amine groups The acid replaces and retains the biological function of the AAV9 capsid.

Also provided herein are AAVrh10 vectors that include artificial genomes that include (i) a polynucleotide-containing expression cassette that is under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has an AAVrh10 capsid protein The amino acid sequence may be at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAVrh10 capsid protein, while retaining the biological function of the AAVrh10capsid capsid. The encoded AAVrh10 capsid may have the sequence of SEQ ID NO: 81 set forth in U.S. Patent No. 9,790,427, which is incorporated by reference in its entirety, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amine groups The acid replaces and retains the biological function of the AAVrh10 capsid.

Gene regulatory elements can be selected to function in mammalian cells (e.g., neurons). The resulting construct contains operably linked components flanked (5' and 3') by functional AAV ITR sequences. Specific examples include vectors derived from AAV serotypes that have tropism for mammalian CNS cells (specifically, neurons) and high transduction efficiency in such cells. A review and comparison of the transduction efficiencies of different serotypes is provided in this patent application. In certain examples, AAV2, AAV5, AAV9, and AAVrh10-based vectors direct long-term expression of polynucleotides in the CNS, for example, by transducing neurons and/or glial cells.

Target polynucleotides flanking the AAV ITR (e.g., encoding inhibitory RNA agents described herein) can be constructed by inserting selected sequences directly into the AAV gene from which the primary AAV open reading frame ("ORF") has been excised. polynucleotide) AAV expression vector. Other portions of the AAV genome can be deleted as long as sufficient portions of the ITR are retained to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published January 23, 1992) and WO 93/03769 (published March 4, 1993). Alternatively, the AAV ITR can be excised from the viral genome or from an AAV vector containing the viral genome using standard nucleic acid ligation techniques and fused to the 5' and 3' ends of a selected nucleic acid construct present in another vector . ITR-containing AAV vectors have been described, for example, in U.S. Patent No. 5,139,941. In particular, several AAV vectors are described and are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226. Additionally, chimeric genes can be synthetically generated to include AAV ITR sequences arranged 5' and 3' relative to one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells can be used, and in some cases, codon optimization of the polynucleotide can be performed by conventional methods. Complete chimeric sequences are assembled from overlapping polynucleotides prepared by standard methods. To produce AAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques such as by transfection. A variety of transfection techniques are well known in the art. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles. .

For example, the specific viral vectors of the present disclosure include, in addition to the nucleic acid sequences of the present disclosure (e.g., SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID In addition to any one of NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, and SEQ ID NO: 238 to SEQ ID NO: 241), it can also Comprises the backbone of an AAV vector plasmid with an ITR derived from an AAV2 virus; a promoter such as a neuronal promoter such as the hSyn promoter and/or the CaMKII promoter, with or without wild-type or mutant forms of WPRE; and rabbit β- Globin polyadenylation sequence (see Table 11 and Table 12). If desired, in the constructs described herein, the hSyn promoter and the CaMKII promoter can be replaced by a constitutive promoter containing a cytomegalovirus enhancer (e.g., CAG or CBA), U6, H1, or 7SK promoter.

The present disclosure further relates to a rAAV comprising (i) an expression cassette containing a polynucleotide that is under the control of a regulatory element and flanked by an ITR, and (ii) an AAV capsid, wherein the polynucleotide encodes an inhibitory RNA (For example, ASO, such as siRNA, shRNA, miRNA or shmiRNA, specifically, an inhibitory RNA having the nucleic acid sequence of any of the following: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229 and SEQ ID NO: 238 to 241 or variants thereof , the variant shares at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62. SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229 and SEQ ID NO: 238 to 241), the inhibitory RNA specifically Binds to at least a portion or region of Grik2 mRNA (e.g., any of the portions or regions of Grik2 mRNA described in: SEQ ID NO: 164 to SEQ ID NO: 193) and inhibits (e.g., knocks down) the cell ( For example, the expression of GluK2 protein in neurons).

AAV vectors may include, for example, inhibitory RNA (eg, siRNA, shRNA, miRNA, or shmiRNA) sequences that bind to Grik2 mRNA, and the hSyn promoter. For example, an AAV vector may contain the nucleic acid sequence of any of: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO : 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229 and SEQ ID NO: 238 to 241 or variants thereof, which have at least the same nucleic acid sequence as any of the following 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Multiple (e.g., 100%)) sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, and SEQ ID NO: 238 to SEQ ID NO: 241; and an hSyn promoter (e.g., having a hSyn promoter of a nucleic acid sequence: SEQ ID NO: 194 to SEQ ID NO: 198 or a variant thereof that shares at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity Sex: SEQ ID NO: 194-198).

Alternatively, the AAV vector may include an inhibitory RNA (eg, siRNA, shRNA, miRNA, or shmiRNA) sequence that binds to Grik2 mRNA and inhibits its expression, as well as the CaMKII promoter. For example, the disclosed AAV vectors may include the nucleic acid sequence of any of the following: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229, SEQ ID NO: 238 to 241, and SEQ ID NO: 258 or a variant thereof, which variant is identical to one of the following Any nucleic acid sequence has at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, SEQ ID NO: 238 to SEQ ID NO: 241 and SEQ ID NO: 258; and CaMKII A promoter (e.g., a CaMKII promoter having a nucleic acid sequence corresponding to any of the following: SEQ ID NO: 199 to SEQ ID NO: 204 or a variant thereof having a nucleic acid sequence corresponding to any of the following) Have at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 199-204). In some embodiments, the AAV vector may include the nucleic acid sequence of any of the following: SEQ ID NO: 4, SEQ ID NO: 19, SEQ ID NO: 34, SEQ ID NO: 135, SEQ ID NO: 141, SEQ ID NO: 147 and SEQ ID NO: 258 or variants thereof, which have at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 4, SEQ ID NO: 19, SEQ ID NO: 34, SEQ ID NO: 135, SEQ ID NO: 141, SEQ ID NO: 147 and SEQ ID NO: 258.

retroviral vector

Delivery vectors used in the methods and compositions described herein can be retroviral vectors. One type of retroviral vectors useful in the methods and compositions described herein are lentiviral vectors. Lentiviral vectors (LVs) are a subset of retroviruses that efficiently transduce a wide range of dividing and non-dividing cell types, conferring stable, long-term expression of polynucleotides. An overview of optimized strategies for packaging and transducing LV is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

The use of lentivirus-based gene transfer technology relies on the in vitro production of recombinant lentiviral particles carrying highly deleted viral genomes that contain target polynucleotides. Specifically, recombinant lentiviruses are recovered by trans co-expression in the following permissive cell lines: (1) packaging constructs, that is, vectors expressing both the Gag-Pol precursor and Rev (or in trans); (2) vectors expressing outer membrane receptors, often of heterologous nature; and (3) The transfer vector consists of viral cDNA with all open reading frames removed, but the sequences required for replication, encapsidation and expression are retained, into which the sequence to be expressed is inserted.

The LV used in the methods and compositions described herein may include 5' long terminal repeats (LTR), HIV message sequence, HIV Psi message 5' splice site (SD), delta-GAG element, Rev response sequence element ( RRE), 3' splice site (SA), elongation factor (EF) 1-α promoter, and 3'-self-inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine segment (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it is a WPRE. The lentiviral vector may further include a pHR' backbone, which may include, for example, as provided below.

Lentigen LV, described in Lu et al., Journal of Gene Medicine 6:963 (2004), can be used to express DNA molecules and/or transduce cells. The LV used in the methods and compositions described herein may include 5' long terminal repeats (LTR), HIV message sequence, HIV Psi message 5' splice site (SD), delta-GAG element, Rev response sequence element ( RRE), 3' splice site (SA), elongation factor (EF) 1-α promoter, and 3'-self-inactivating L TR (SIN-LTR). As appropriate, one or more of these areas is replaced by another area that performs a similar function.

Enhancer elements can be used to increase the expression of modified DNA molecules or to increase lentiviral integration efficiency. LVs used in the methods and compositions described herein may include nef sequences. LVs used in the methods and compositions described herein may include cPPT sequences that enhance vector integration. cPPT serves as a second starting point for (+) strand DNA synthesis and introduces partial strand overlap in the middle of its native HIV genome. Introduction of cPPT sequences into the transfer vector backbone greatly increases the amount of nuclear transport and integration into target cell DNA. LVs used in the methods and compositions described herein may include woodchuck post-transcriptional regulatory elements (WPRE). WPRE acts at the transcriptional level by promoting nuclear export of transcripts and/or by increasing polyadenylation efficiency of nascent transcripts, thereby increasing the total amount of mRNA in the cell. Addition of WPRE to LV resulted in significantly enhanced expression levels of polynucleotides from several different promoters in vitro and in vivo. LVs used in the methods and compositions described herein can include both cPPT sequences and WPRE sequences. Vectors may also include IRES sequences that allow expression of multiple polypeptides from a single promoter.

In addition to IRES sequences, other elements that allow the expression of multiple polynucleotides may also be useful. Vectors used in the methods and compositions described herein may include multiple promoters that allow expression of more than one polynucleotide. Additional elements that allow expression of multiple polynucleotides identified in the future are useful and can be used in vectors suitable for use with the compositions and methods described herein. Carriers used in the methods and compositions described herein can be clinical grade carriers.

Accordingly, retroviral vectors may be used in conjunction with the disclosed methods and compositions. Retroviruses can be chosen as gene delivery vectors because they can integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specific cell strains. To construct retroviral vectors, nucleic acid encoding a gene of interest is inserted into the viral genome in place of specific viral sequences to produce a replication-deficient virus. To produce virions, packaging cell lines were constructed containing gag, pol and/or env genes but without LTR and/or packaging components. When a recombinant plasmid containing cDNA is introduced into this cell line along with the retroviral LTR and packaging sequence (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted. into the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a variety of cell types.

Additionally, lentiviral vectors can be used in conjunction with the methods and compositions disclosed herein. Accordingly, one object of the present disclosure relates to an inhibitory RNA (e.g., siRNA, shRNA, miRNA, or shmiRNA) sequence (e.g., any of the inhibitory RNA sequences described in: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO : 229, SEQ ID NO: 238 to SEQ ID NO: 241 and SEQ ID NO: 258), the lentiviral vector binds to Grik2 mRNA and inhibits its expression.

Accordingly, the lentiviral vector may include the nucleic acid sequence of any one of: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229, SEQ ID NO: 238 to 241, and SEQ ID NO: 258 or a variant thereof, which variant is consistent with any of the following One has at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, SEQ ID NO: 238 to SEQ ID NO: 241 and SEQ ID NO: 258. Lentiviral vectors can include inhibitory RNA sequences (eg, siRNA, shRNA, miRNA, or shmiRNA) that bind to Grik2 mRNA and inhibit its expression, as well as the hSyn promoter.

Lentiviral vectors can include, for example, inhibitory RNA (eg, siRNA, shRNA, miRNA, or shmiRNA) sequences that bind to Grik2 mRNA, and the hSyn promoter. For example, a lentiviral vector may contain the nucleic acid sequence of any one of: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229, SEQ ID NO: 238 to 241 and SEQ ID NO: 258 to SEQ ID NO: 260 or variants thereof, which The nucleic acid sequence has at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, SEQ ID NO: 238 to SEQ ID NO: 241 and SEQ ID NO : 258 to 260; and an hSyn promoter (e.g., an hSyn promoter having a nucleic acid sequence consistent with any of the following: SEQ ID NO: 194 to SEQ ID NO: 198 or a variant thereof that is consistent with the following) Any of the nucleic acid sequences has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 194-198).

Alternatively, the lentiviral vector may include an inhibitory RNA (eg, siRNA, shRNA, miRNA, or shmiRNA) sequence that binds to Grik2 mRNA and inhibits its expression, as well as the CaMKII promoter. For example, the disclosed lentiviral vectors can include the nucleic acid sequence of any one of: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to 229, SEQ ID NO: 238 to 241 and SEQ ID NO: 258 to SEQ ID NO: 260 or variants thereof, The variant shares a nucleic acid sequence identity of at least 85% with any of the following (at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62 , SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229, SEQ ID NO: 238 to SEQ ID NO: 241 and SEQ ID NO: 258 to 260; and a CaMKII promoter (e.g., a CaMKII promoter having a nucleic acid sequence corresponding to any of: SEQ ID NO: 199 to SEQ ID NO: 204 or a variant thereof, which variant is identical to The nucleic acid sequences have at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 199-204).

Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, also contain other genes with regulatory or structural functions. Greater complexity enables the virus to regulate its life cycle, just as it does during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuating HIV virulence genes, such as deletion of the genes env, vif, vpr, vpu, and nef, thereby rendering the vector biologically safe. Lentiviral vectors are known in the art, see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. Generally, vectors are plasmid-based or viral-based and are configured to carry the necessary sequences for the incorporation of foreign nucleic acids as well as for the selection and transfer of nucleic acids into host cells. Targeting vectors to gag, pol and env genes are also known in the art. Therefore, the relevant genes are selected and cloned into the vector of choice and then used to transform the target cells of interest. Recombinant lentiviruses capable of infecting non-dividing cells, in which a suitable host cell is transfected with two or more vectors carrying packaging proteins (i.e., gag, pol, and env) as well as rev and tat, are described in U.S. Patent No. 5,994,136 , which is incorporated herein by reference. This disclosure provides a first vector that provides nucleic acid encoding viral gag and pol genes and a second vector that provides nucleic acid encoding viral env to generate packaging cells. A vector providing a heterologous gene is introduced into the packaging cell to obtain a producer cell, which releases infectious virus particles carrying the target foreign gene. env may be an amphiphilic outer membrane protein that allows transduction of cells of humans and other species. Generally, the nucleic acid molecules or vectors of the present disclosure include "control sequences", which collectively refer to promoter sequences, polyadenylation message sequences, transcription termination sequences, upstream regulatory domains, origins of replication, and internal ribosome entry sites ("IRES"). ), enhancers, etc., which together provide the replication, transcription and translation of coding sequences in recipient cells. Not all such control sequences need be present at all times, as long as the selected coding sequence is capable of replication, transcription and translation in the appropriate host cell.

Nucleic acid vectors that exhibit tropism for specific target cells such as hippocampal neurons, e.g., DGCs, can be used to deliver inhibitory polynucleotides described herein.

viral regulatory elements

Viral regulatory elements are components of delivery vectors used to introduce nucleic acid molecules into host cells. Viral regulatory elements are optionally retroviral regulatory elements. For example, viral regulatory elements can be the LTR and gag sequences from HSC1 or MSCV. Retroviral regulatory elements can be derived from lentiviruses, or they can be heterologous sequences identified from other genome regions. As other viral regulatory elements become known, such viral regulatory elements may be used in conjunction with the methods and compositions described herein.

encoding Grik2 Viral vectors of inhibitory polynucleotides

The present disclosure relates to nucleic acid vectors for delivering heterologous polynucleotides, wherein the polynucleotides encode inhibitory RNA agents (e.g., siRNA, shRNA, miRNA or shmiRNA) construct. Accordingly, one object of the present disclosure is to provide a vector comprising an inhibitory polynucleotide sequence that is associated with at least one region or portion of Grik2 mRNA (e.g., a region of Grik2 mRNA selected from any of the following or Any of the following: SEQ ID NO: 164 to SEQ ID NO: 193 or a variant thereof that is at least 85% identical to any of the following (e.g., at least 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more (eg, 100%)) Sequence identity: SEQ ID NO: 164 to SEQ ID NO: 193) Complete or substantially complementary. Vectors of the present disclosure may include any variant of an inhibitory polynucleotide sequence that is completely or substantially complementary to one or more regions of Grik2 mRNA. Furthermore, vectors of the present disclosure may include any variant of an inhibitory polynucleotide sequence that is completely or substantially complementary to Grik2 mRNA encoding any variant of GluK2 protein.

Accordingly, DNA encoding the double-stranded RNA of interest is incorporated into a gene cassette, such as an expression cassette, where transcription of the DNA is controlled by a promoter and/or other regulatory elements. Incorporation of DNA expressing target Grik2 inhibitory RNA (e.g., SEQ ID NO: 1 to 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to SEQ ID NO: 229, SEQ ID NO: 238 to SEQ ID NO: 241, SEQ ID NO: 250 to SEQ ID NO: 251 and SEQ ID NO: 256 to SEQ ID NO: 261) and are encapsulated by the target viral vector for delivery to the target cells. Thus, the viral vectors of the present disclosure encode antisense RNA that hybridizes to any Grik2 mRNA transcript isoform (eg, any of SEQ ID NO: 164 to SEQ ID NO: 174). The viral vector encodes, for example, any of the inhibitory polynucleotides listed in Tables 2, 4, 6, and 8.

Vectors of the present disclosure deliver polynucleotides encoding inhibitory RNAs that recognize or bind to at least a portion or region of Grik2 mRNA (e.g., any of the regions or portions of Grik2 mRNA described in: SEQ ID NO: 164 to SEQ ID NO: 193 or a variant thereof that shares at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%) the nucleic acid sequence of any of the following , 98%, 99% or more (e.g., 100%)) sequence identity: SEQ ID NO: 164-193). The heterologous polynucleotide encoding an inhibitory RNA agent can be part of a larger construct or scaffold that ensures the expression of such inhibitory RNA in cells (e.g., mammalian cells, such as human cells, such as neurons Processing in cells such as DGCs or glutamatergic pyramidal neurons. Polynucleotides encoding any of the siRNAs listed in Tables 2 to 9 may include microRNA genes (eg, E-miR-30, E-miR-218-1, or E-miR-124-3, etc.) Precursor or part, such as 5' flanking sequence, 3' flanking sequence or loop sequence of a microRNA gene. In some embodiments, a polynucleotide encoding any of the siRNAs listed in Tables 2 to 9 may include one or more microRNA genes (e.g., E-miR-30, E-miR-218-1 or E-miR-124-3) precursor or part. In some embodiments, the polynucleotide may include precursors or portions of two or more microRNA genes (e.g., E-miR-30, E-miR-218-1, or E-miR-124-3) . In preferred embodiments, the polynucleotide may include precursors or portions of E-miR-30 and E-miR-218-1.

Accordingly, one object of the present disclosure relates to an expression vector comprising a heterologous polynucleotide and containing: from 5' to 3', e.g., a promoter (e.g., any of the promoters described in Table 11) , an optional intron (e.g., any of the introns described in Table 12), a nucleotide sequence encoding an inhibitory RNA agent that inhibits the expression of Grik2 mRNA (e.g., an inhibitory RNA agent that A nucleic acid sequence having any of the following: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO : 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261 or variants thereof, which variants are consistent with the following The nucleic acid sequence of any one of the nucleic acid sequences has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251, and SEQ ID NO: 256 to 261), and polyadenylation sequences (e.g., the polyadenylation sequences listed in Table 12 any one in the sequence). The expression vector may also include: from the 5' inverted terminal repeat (ITR) to the 3' ITR, the 5' ITR (e.g., either of the 5' or 3' ITR sequences described in Table 12), a promoter, Optionally, the intron, the nucleotide sequence encoding the inhibitory RNA that inhibits the expression of Grik2 mRNA, the polynucleotide sequence and the 3' ITR are selected. The expression vector may further contain spacer and/or linker sequences joined to any of the aforementioned vector elements.

In specific examples, the expression vector or polynucleotide may include nucleotide sequences encoding stems and loops forming a stem-loop structure, wherein the loops include encoding any of the inhibitory RNA agents listed in Tables 2-9 For example, the expression vector or polynucleotide may include a nucleic acid sequence encoding a loop region, wherein the loop region may be derived in whole or in part from a wild-type microRNA sequence gene (e.g., E-miR-30, E -miR-218-1 or E-miR-124-3, etc.) or completely artificial. In specific examples, the loop region can be an E-miR-30a loop sequence. In some embodiments, expression vectors or polynucleotides can include nucleic acid sequences encoding two loop regions, wherein the loop regions can be derived in whole or in part from wild-type microRNA sequences (e.g., E-miR-30, E -miR-218-1 or E-miR-124-3). In specific embodiments, the loop region may include an E-miR-30 loop region and an E-miR-218-1 loop region.

Additionally, the one or more stem-loop structures may include a guide sequence (e.g., an antisense RNA sequence, such as any of the following: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) and follower sequences (e.g., any of the following: SEQ ID NO: 31 to SEQ ID NO: 45, SEQ ID NO: 80 to SEQ ID NO: 96, SEQ ID NO: 121 to SEQ ID NO: 132 , SEQ ID NO: 145 to SEQ ID NO: 150, SEQ ID NO: 234 to 237, and SEQ ID NO: 246 to SEQ ID NO: 249), the follower sequence is complementary to all or part of the leader sequence. For example, the follower sequence may be complementary to all but 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide of the leader sequence, or the follower sequence may be complementary to Any of the following are complementary: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In a specific example, one stem-loop structure may include the leader sequence of SEQ ID NO: 19 and the follower sequence of SEQ ID NO: 34, and a second stem-loop structure may include the leader sequence of SEQ ID NO: 141 and the follower sequence of SEQ ID NO: 147. Any sequence variants of these four sequences (SEQ ID NO: 19, SEQ ID NO: 34, SEQ ID NO: 141 and SEQ ID NO: 147) can be included in the stem-loop structure. These sequence variants Have at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%)) sequence identity to any of the following: SEQ ID NO: 19. SEQ ID NO: 34, SEQ ID NO: 141 and SEQ ID NO: 147. In some embodiments, the one or more stem-loop structures may comprise a nucleic acid sequence that is at least 85% (e.g., at least 90%, 95%, 99%, or more (e.g., 100%) identical to any of )) Sequences with sequence identity: SEQ ID NO: 4, SEQ ID NO: 135 and SEQ ID NO: 258.

Pre-miRNA or pri-miRNA scaffolds include guide (i.e., antisense) sequences of the present disclosure. pri-miRNA scaffolds include pre-miRNA scaffolds, and pri-miRNA can be 50 to 800 nucleotides in length (e.g., 50 to 800, 75 to 700, 100 to 600, 150 to 500, 200 to 400, or 250 nucleotides). to 300 nucleotides). In specific examples, pri-mRNA can be 50 to 100 nucleotides (e.g., between 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 nucleotides), 100 to 200 nucleotides (e.g., between 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, or 190 to 200 nucleotides between nucleotides), 200 to 300 nucleotides (for example, between 200 to 210, 210 to 220, 220 to 230, 230 to 240, 240 to 250, 250 to 260, 260 to 270, 270 to 280, 280 to 290, or 290 to 300 nucleotides), 300 to 400 nucleotides (e.g., between 300 to 310, 310 to 320, 320 to 330, 330 to 340, 340 to 350, 350 to 360, 360 to 370, 370 to 380, 380 to 390, or 390 to 400 nucleotides), 400 to 500 nucleotides (e.g., between 400 to 410, 410 to 420, 420 to 430, 430 to 440, 440 to 450, 450 to 460, 460 to 470, 470 to 480, 480 to 490, or 490 to 500 nucleotides), 500 to 600 nucleotides (e.g., by 500 to 510, 510 to 520, 520 to 530, 530 to 540, 540 to 550, 550 to 560, 560 to 570, 570 to 580, 580 to 590, or 590 to 600 nucleotides), 600 to 700 Nucleotides (e.g., between 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 680 to 690, or 690 to 700 nucleotides between nucleotides), or 700 to 800 nucleotides (e.g., between 700 to 710, 710 to 720, 720 to 730, 730 to 740, 740 to 750, 750 to 760, 760 to 770, 770 to 780 , 780 to 790, or 790 to 800 nucleotides). These engineered scaffolds allow processing of pre-miRNA into double-stranded RNA containing leader and follower strands. Thus, pre-miRNA includes: the 5' arm, which includes the sequence encoding the guide (i.e., antisense sequence) RNA; the loop sequence, which is typically derived from wild-type miRNA (e.g., E-miR-30, E-miR-218 -1 or E-miR-124-3, etc.); and the 3' arm, which includes a sequence encoding a follower strand that is completely or substantially complementary to the leader strand (ie, the sense sequence). Pre-miRNA "stem-loop" structures are typically longer than 50 nucleotides, for example, 50 to 150 nucleotides in length (e.g., 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, or 140 to 150 nucleotides), 50 to 110 nucleotides (e.g., 50 to 60, 60 to 70, 70 to 80, 80 to 90 , 90 to 100, 100 to 110 nucleotides), or 50 to 80 nucleotides (e.g., 50 to 60, 60 to 70, 70 to 80 nucleotides). Pri-miRNA further includes 5' flanking and 3' flanking sequences flanking the 5' and 3' arms respectively. The flanking sequences are not necessarily contiguous with other sequences (arm regions or guide sequences), are unstructured, unpaired regions, and can also be derived in whole or in part from one or more wild-type pri-miRNA scaffolds (e.g., pri-miRNA The miRNA scaffold is derived in whole or in part from one or more of E-miR-30, E-miR-218-1 or E-miR-124-3, etc.). The flanking sequences are each at least 4 nucleotides in length, or up to 300 nucleotides or more (e.g., 4 to 300, 10 to 275, 20 to 250, 30 to 225, 40 to 200, 50 to 175 , 60 to 150, 70 to 125, 80 to 100 or 90 to 95 nucleotides). Spacer sequences can be inserted between the preceding sequence structures and in most cases provide linking polynucleotides, e.g., 1 to 30 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides), to provide flexibility to the entire pre-miRNA structure without interfering with function. The spacer may be derived from a naturally occurring linking group from naturally occurring RNA, a portion of a naturally occurring linking group, a polyadenylate or polyuridylate, or a random sequence of nucleotides, so long as the spacer is neither Interference with double-stranded RNA processing does not interfere with the binding/interaction of the guide RNA with the target mRNA sequence.

According to the methods and compositions disclosed herein, an expression vector or polynucleotide including a nucleotide sequence may further encode (i) a 5' stem-loop arm, which includes a guide (e.g., antisense) strand and optionally a 5' a spacer sequence; and (ii) a 3' stem-loop arm, which includes a follower (eg, sense) strand and optionally a 3' spacer sequence. In another example, an expression vector or polynucleotide comprising a nucleotide sequence may further encode (i) a 5' stem-loop arm including a follower strand and optionally a 5' spacer sequence; and (ii) 3 'Stem-loop arm, which includes the guide strand and optionally the 3' spacer sequence. In another example, a uridine wobble base is present at the 5' end of the leading strand. In yet another example, the expression vector or polynucleotide includes a leader 5' flanking region located upstream of the leader sequence, and the flanking region can be of any length and can be derived in whole or in part from a wild-type microRNA sequence, which can be heterologous. or may be derived from a different source than the other flanking regions or loops of the miRNA, or may be completely artificial. The size and origin of the 3' flanking region can be a mirror image of the 5' flanking region, and the 3' flanking region can be located downstream (i.e., 3') of the leader sequence. In yet another example, one or both of the 5' flanking sequence and the 3' flanking sequence are absent.

The expression vector or polynucleotide may include a nucleotide sequence further encoding a first flanking region (e.g., any of the 5' flanking regions described in Table 8) that includes the 5' flanking sequence and Optional 5' spacer sequence. In certain instances, the first flanking region is located upstream (ie, 5') of the follower strand. In another example, an expression vector or polynucleotide comprising a nucleotide sequence encodes a second flanking region (e.g., any of the 3' flanking regions described in Table 8), the second flanking region comprising 3 'Flanking sequences and optional 3' spacer sequences. In a specific example, the first flanking region is located 5' from the leading strand.

According to the methods and compositions disclosed herein, an expression vector or polynucleotide may comprise at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%) of any of the following: , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity of the nucleic acid sequence: SEQ ID NO: 1 to SEQ ID NO: 34. SEQ ID NO: 135 to SEQ ID NO: 147, SEQ ID NO: 226 to 229 or SEQ ID NO: 256. In specific examples, the expression vector or polynucleotide can include a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 256. In some embodiments, the expression vector or polynucleotide may comprise the following nucleic acid sequence: SEQ ID NO: 256. In another specific example, the expression vector or polynucleotide may comprise a sequence having the following properties: At least 85% (for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Nucleic acid sequences with multiple (eg, 100%) identity: SEQ ID NO: 1 to SEQ ID NO: 34 and SEQ ID NO: 135 to SEQ ID NO: 147. In another example, the expression vector or polynucleotide can comprise at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, Nucleic acid sequences that are 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identical: SEQ ID NO: 46-62. In another example, the expression vector or polynucleotide can comprise at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, Nucleic acid sequences with 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity: SEQ ID NO: 97-108. In yet another example, the expression vector or polynucleotide can comprise at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, Nucleic acid sequences with 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity: SEQ ID NO: 133-138.

In another example, the expression vector or polynucleotide includes a nucleotide sequence encoding: (a) Stem-loop sequence, which includes, from 5' to 3': (i) A 5' stem-loop arm that includes a follower nucleotide sequence that is complementary or substantially complementary to the leader nucleotide sequence (such as having a sequence that is at least 85% (e.g., at least 86%, 87%) identical to any of the following) , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity of the nucleic acid Sequences following the sequence: SEQ ID NO: 31 to SEQ ID NO: 45, SEQ ID NO: 80 to SEQ ID NO: 96, SEQ ID NO: 121 to SEQ ID NO: 132, SEQ ID NO: 145 to SEQ ID NO : 150, SEQ ID NO: 234 to 237 and SEQ ID NO: 246 to 249); (ii) A microRNA loop region, wherein the loop region includes a microRNA loop sequence (e.g., E-miR-30a, miR-218-1 or E-miR-124-3 loop sequence (e.g., having a sequence selected from MicroRNA sequence of any one of the nucleic acids: SEQ ID NO: 4 to SEQ ID NO: 225); (iii) a 3' stem-loop arm that includes a guide nucleotide sequence (such as having a sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%) identical to any of the following) %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity of the nucleic acid sequence: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to 233 and SEQ ID NO: 242 to 245); (b) The 5' flanking region located 5' of the follower strand (e.g., any of the 5' flanking regions described in Table 13); and (c) A 3' flanking region located 3' of the guide strand (e.g., any of the 3' flanking regions described in Table 13), where the second flanking region includes the 3' flanking sequence and optionally the 3' flanking region spacer sequence. In some embodiments, the expression vector or polynucleotide includes a nucleic acid sequence encoding: (a) a stem-loop sequence including, from 5' to 3': (i) A 5' stem-loop arm that includes a follower nucleotide sequence that is complementary or substantially complementary to the leader nucleotide sequence (such as having a sequence that is at least 85% (e.g., at least 86%, 87%) identical to any of the following) , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) identity of the nucleic acid Sequences following the sequence: SEQ ID NO: 34 and SEQ ID NO: 147); (ii) a microRNA loop region, wherein the loop region includes a microRNA loop sequence (e.g., E-miR-30a or miR-218-1 loop Sequence (e.g., a microRNA sequence having a nucleic acid selected from any of: SEQ ID NO: 4 or SEQ ID NO: 135); (iii) a 3' stem-loop arm including a guide nucleotide sequence ( Such as having at least 85% of any of the following (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, A primer sequence for a nucleic acid sequence with 97%, 98%, 99% or more (eg, 100%) sequence identity: SEQ ID NO: 19 and SEQ ID NO: 141). (b) The 5' flanking region located 5' of the follower strand (e.g., any of the 5' flanking regions described in Table 13); and (c) A 3' flanking region located 3' of the guide strand (e.g., any of the 3' flanking regions described in Table 13), where the second flanking region includes the 3' flanking sequence and optionally the 3' flanking region spacer sequence. In some embodiments, the expression vector or polynucleotide includes a nucleic acid sequence that includes two stem-loop sequences, wherein (a) one stem-loop sequence contains the following leader sequence: SEQ ID NO: 19 or a variant thereof, The variant is at least 85% identical to SEQ ID NO: 19 (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; microRNA loop region: SEQ ID NO: 4 or a variant thereof that 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; and the following sequence: SEQ ID NO: 34 or a variant thereof that is 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; and (b) The second stem-loop sequence contains the following leader sequence: SEQ ID NO: 141 or a variant thereof that is 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; microRNA Loop region: SEQ ID NO: 135 or a variant thereof that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%) identical to SEQ ID NO: 135 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity; and the following sequence: SEQ ID NO: 147 or a variant thereof , the variant is at least 85% identical to SEQ ID NO: 135 (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; 147. In a preferred embodiment, the expression vector or polynucleotide includes a nucleic acid sequence comprising two stems Loop sequence, wherein (a) one stem-loop sequence contains the leader sequence of SEQ ID NO: 19, the microRNA loop region of SEQ ID NO: 4, and the follower sequence of SEQ ID NO: 34, and (b) the second stem-loop sequence The sequence includes the leader sequence of SEQ ID NO: 141, the microRNA loop region of SEQ ID NO: 135, and SEQ ID NO: 147. In some embodiments, the expression vector or polynucleotide includes at least SEQ ID NO: 256. 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 of the nucleotide sequence. In some embodiments, the expression vector or polynucleotide has the nucleotide sequence of SEQ ID NO: 256.

In another example, the expression vector or polynucleotide includes a nucleotide sequence encoding: (a) Stem-loop sequence, which includes, from 5' to 3': (i) A 5' stem-loop arm that includes a guide nucleotide sequence (such as having a sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%) identical to any of the following) %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity to a nucleic acid sequence leader sequence: SEQ ID NO: 16 To SEQ ID NO: 30, SEQ ID NO: 63 To SEQ ID NO: 79, SEQ ID NO: 109 To SEQ ID NO: 120, SEQ ID NO: 139 To SEQ ID NO: 144, SEQ ID NO: 230 to 233 and SEQ ID NO: 242 to 245); (ii) A microRNA loop region, wherein the loop region includes a microRNA loop sequence (e.g., E-miR-30a, miR-218-1 or E-miR-124-3 loop sequence (e.g., having a sequence selected from The microRNA sequence of any of the nucleic acids: SEQ ID NO: 219, SEQ ID NO: 222 or SEQ ID NO: 225); (iii) a 3' stem-loop arm that includes a follower nucleotide sequence that is complementary or substantially complementary to the leader nucleotide sequence (such as having a sequence that is at least 85% (e.g., at least 86%, 87%) identical to any of the following) , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) sequence identity Follower sequences of nucleic acid sequences: SEQ ID NO: 31 to SEQ ID NO: 45, SEQ ID NO: 80 to SEQ ID NO: 96, SEQ ID NO: 121 to SEQ ID NO: 132, SEQ ID NO: 145 to SEQ ID NO: 150, SEQ ID NO: 234 to 237 and SEQ ID NO: 246 to 249); (b) The 5' flanking region located at 5' of the guide strand (e.g., any of the 5' flanking regions described in Table 13); and (c) A 3' flanking region located at 3' of the follower strand (e.g., any of the 3' flanking regions described in Table 13), wherein the second flanking region includes the 3' flanking sequence and optionally the 3' flanking region spacer sequence.

The length of the aforementioned leading and following strands may be 19 to 50 (for example, 19, 20, 21, 22, 23, 24, 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45 or 46 to 50) length between nucleotides. In a specific example, the guide strand is 19 nucleotides in length. In another example, the guide strand is 20 nucleotides in length. In another example, the guide strand is 21 nucleotides in length. In another example, the guide strand is 22 nucleotides in length. In another example, the guide strand is 23 nucleotides in length. In another example, the guide strand is 24 nucleotides in length. In another example, the guide strand is 25 nucleotides in length. In another example, the guide strand is 26 to 30 nucleotides in length. In another example, the guide strand is 31 to 35 nucleotides in length. In another example, the guide strand is 36 to 40 nucleotides in length. In another example, the guide strand is 41 to 45 nucleotides in length. In another example, the guide strand is 46 to 50 nucleotides in length. In a specific example, the follower strand is 19 nucleotides in length. In another example, the follower strand is 20 nucleotides in length. In another example, the follower strand is 21 nucleotides in length. In another example, the follower strand is 22 nucleotides in length. In another example, the follower strand is 23 nucleotides in length. In another example, the follower strand is 24 nucleotides in length. In another example, the follower strand is 25 nucleotides in length. In another example, the follower strand is 26 to 30 nucleotides in length. In another example, the follower strand is 31 to 35 nucleotides in length. In another example, the follower strand is 36 to 40 nucleotides in length. In another example, the follower strand is 41 to 45 nucleotides in length. In another example, the follower strand is 46 to 50 nucleotides in length.

The length of the leader and follower sequences can vary between the miRNA scaffolds into which the leader and follower strands are incorporated. When a given guide is adapted within a miRNA scaffold, the length of the guide can be extended to accommodate the natural structure and processing of a given miRNA scaffold. For example, guide sequences generated from E-miR-30 scaffolds are typically 22 nucleotides long. For most scaffolds, the leader sequence is extended at the 3' end to be additionally complementary to the target mRNA sequence, but this may involve modification of the 5' start site of the leader, depending on the sequence of the miRNA scaffold.

In some cases, it may be necessary to modify the levels of expression and/or processing patterns of the miRNA guide and follower strands to enhance or modify the targeting ability of a given construct. Therefore, within a given miRNA framework/scaffold, the positions of the leader and follower strands are interchangeable; this may be in the context of a design that includes a filler sequence (e.g., SEQ ID NO: 250 or SEQ ID NO: 251), or Possibly in situations where the design has no filler. This may additionally be in the context of double constructs or polyplexes. To accommodate this change, the sequence of leading and/or following strands can be modified from the template "parent" design. Alternatively, the lead and/or follower sequences may be modified to effect changes in lead and follower performance and/or processing patterns.

In a specific example, the vector or polynucleotide includes an E-miR-30a sequence, wherein the 5' flanking region includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 217 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In the following examples, the vector or polynucleotide includes an E-miR-30a sequence, wherein the 3' flanking region includes a nucleotide sequence that is at least 90% (e.g., at least 90%) identical to SEQ ID NO: 218 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In another example, the vector or polynucleotide includes an E-miR-30a structure, wherein the loop region includes the nucleotide sequence of SEQ ID NO: 219, or is at least 90% (e.g., at least 90%) identical to SEQ ID NO: 219 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In a specific example, the vector or polynucleotide includes a miR-218-1 sequence, wherein the 5' flanking region includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 220 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In the following examples, the vector or polynucleotide includes a miR-218-1 sequence, wherein the 3' flanking region includes a nucleotide sequence that is at least 90% (e.g., at least 90%) identical to SEQ ID NO: 221 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In another example, the vector or polynucleotide includes a miR-218-1 structure, wherein the loop region includes the nucleotide sequence of SEQ ID NO: 222, or is at least 90% (e.g., at least 90%) identical to SEQ ID NO: 219 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In a specific example, the vector or polynucleotide includes an E-miR-124-3 sequence, wherein the 5' flanking region includes a nucleotide sequence that is at least 90% (e.g., at least 90%) identical to SEQ ID NO: 223 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In the following examples, the vector or polynucleotide includes an E-miR-124-3 sequence, wherein the 3' flanking region includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 224 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

In another example, the vector or polynucleotide includes an E-miR-124-3 structure, wherein the loop region includes the nucleotide sequence of SEQ ID NO: 225, or is at least 90% identical to SEQ ID NO: 225 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) the same (see Table 13).

The expression vector may be a plasmid and may include, for example, one or more of the following: an intron sequence (e.g., an intron sequence of: SEQ ID NO: 205 or SEQ ID NO: 206 or a variant thereof, the A variant is at least 85% identical (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the following nucleic acid sequence (e.g., 100%)) Sequence identity: SEQ ID NO: 205 or SEQ ID NO: 206), linker sequence, or filler sequence (e.g., SEQ ID NO: 250 or SEQ ID NO: 251). Table 13. MicroRNA sequences _ instruction SEQ ID NO Nucleotide sequence E-hsa-miR-30a 5' flanking region 217 gtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttggaaacacttgctgggattacttcttcaggttaacccaacagaaggctAAagaaggtatattgctgttgacagtgagcgac E-hsa-miR-30a 3' flanking region 218 gctgcctactgcctcggaCttcaaggggctactttaggagcaattatcttgtttaactaaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtataaatta E-hsa-miR-30a loop region 219 ctgtgaagccacagatggg E-hsa-miR-218-1 5' flanking region 220 acgtttccagaacgtctgtagcttttctcctccttccctccattttcctcttggtcttacctttggcctagtggttggtgtagtgataatgtagcgagattttctg E-hsa-miR-218-1 3' flanking region 221 tggaacgtcacgcagctttctacagcatgacaagctgctgaggcttaaatcaggattttcctgtctctttctacaaaatcaaaatgaaaaaagagggctttttaggcatctccgagattatgtg E-hsa-miR-218-1 loop region 222 ggttgcgaggtatgagtaaa E-hsa-miR-124-3 5' flanking region 223 tctgccgcggaaaggggagaagtgtgggctcctccgagtcgggggcggactgggacagcacagtcggctgagcgcagcgccccccgccctgcccgccacgcggcgaagacgcctgagcgttcgcgcccctcgggcgaggacccccacgcaagcccgagccggtcccgaccctggccccgacgctcgccg cccgccccagccctgagggcccctc E-hsa-miR-124-3 3' flanking region 224 gagaggcgcctccgccgctcctttctcatggaaatggcccgcgagcccgtccggcccagcgcccctcccgcgggaggaaggcgagcccgcccccggcggccattcgcgccgcggacaaatccggcgaacaatgcgccgcccagagtgcggcccagctgccgggccggggatctggccgcgggacacaaaagg ggcccgcacgcctctggcgt E-hsa-miR-124-3 loop region 225 atttaatgtctatacaat

Accordingly, one object of the present disclosure relates to a vector comprising a polynucleotide having a nucleic acid sequence of any of the following: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261 or a variant thereof that shares at least 85% (at least 85%, 86%, 87%, 88%, 89%, 90%) the nucleic acid sequence of any of the following %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251, and SEQ ID NO: 256 to 261. For example, a vector can include a polynucleotide that is at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more) identical to any of the following nucleic acid sequences: Multiple (e.g., 100%)) sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261. In another example, a vector can include a polynucleotide that shares at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or More (e.g., 100%)) Sequence identity: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261. The vector of the present disclosure may further comprise a polynucleotide having a nucleic acid sequence of any of the following: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261.

Specifically, the vector may include: the sequence of any one of: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NO: 256 to 261 or variations thereof A variant that shares 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: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62. SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 208 to 229, SEQ ID NO: 238 to 241, SEQ ID NO: 250 to 251 and SEQ ID NOs: 256 to 261; and a promoter (eg, any of the promoters listed in Table 11).

Variants discussed above may include, for example, naturally occurring variants due to allelic variation between individuals (e.g., polymorphisms), alternative splicing forms, etc. The term variant also includes genetic sequences of the present disclosure from other sources or organisms. Variants can be substantially homologous to sequences according to the present disclosure. Variants of the genes of the present disclosure also include nucleic acid sequences that hybridize under stringent hybridization conditions to the sequence defined above (or its complement). Typical stringent hybridization conditions include temperatures above 30°C, above 35°C, or above 42°C, and/or salinities below approximately 500 mM or below 200 mM. Hybridization conditions can be adjusted by, for example, changing the temperature, salinity and/or concentration of other reagents such as SDS, SSC, etc.

The present disclosure further provides non-viral vectors (eg, plasmids containing polynucleotides encoding Grik2- targeting inhibitory RNA agents disclosed herein) for delivering heterologous polynucleotides to target cells of interest. In other cases, the viral vectors of the present disclosure may be AAV vectors, adenovirus, retrovirus, lentivirus or herpes virus vectors.

One or more presentation cassettes can be used. Each expression cassette can include at least one promoter sequence (e.g., a neuronal cell promoter) operably linked to a sequence encoding the target RNA. Each expression cassette can be composed of additional regulatory elements, spacers, introns, UTRs, polyadenylation sites, etc. Expression cassettes may be polycistronic with respect to polynucleotides encoding, for example, two or more inhibitory RNA agents. The expression cassette may further include a promoter, nucleic acid encoding one or more inhibitory RNA agents of interest, and polyadenylation sequences. In specific examples, the expression cassette includes a 5'-promoter sequence, a polynucleotide sequence encoding a first inhibitory RNA agent of interest (e.g., any of the following: SEQ ID NO: 16 to SEQ ID NO: 30 , SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245), a sequence encoding a second inhibitory RNA agent of interest (e.g., any of the following: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245) and polyadenylation sequence -3'. In a preferred embodiment, the performance cassette includes a sequence that is 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: SEQ ID NO: 256. In some embodiments, the performance cassette has SEQ ID NO: 256 sequence. In a preferred embodiment, the performance cassette includes a sequence that is 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: SEQ ID NO: 257. In some embodiments, the performance cassette has the sequence of SEQ ID NO: 257.

The viral vector may further include genes encoding antibiotic resistance (such as AmpR, conmycin, hygromycin B, geneticin, blasticidin S, oxymycin only, carbencillin, chloramphenicol, Nourseothricin or puromycin resistance gene) nucleic acid sequence. Example presentation box

The present disclosure provides expression cassettes that, when incorporated into an expression vector (eg, a plasmid or a viral vector (eg, an AAV or a lentiviral vector)), facilitate encoding of an inhibitory RNA agent (eg, having any of the following Inhibitory RNA agents of nucleic acid sequences: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to 120, SEQ ID NO: 139 to SEQ ID NO : 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245), the inhibitory RNA agent hybridizes to Grik2 mRNA and inhibits its expression. Typically, expression cassettes incorporated into nucleic acid vectors will include heterologous polynucleotides containing heterologous gene regulatory sequences (e.g., a promoter (e.g., any of the promoters described in Table 11 a) and optional enhancer sequences (e.g., the enhancer sequences described in Table 12), 5' flanking sequences (e.g., the 5' flanking sequences described in Table 13), stem-loop 5' arms The stem-loop sequence, loop sequence (for example, the microRNA loop sequence described in Table 13), stem-loop 3' arm, and 3' flanking sequence (for example, the 3' flanking sequence described in Table 13) are selected as appropriate. The woodchuck hepatitis post-transcriptional regulatory element (WRPE) and polyadenylation sequence (e.g., SEQ ID NO: 213-216). In the case of AAV vectors, the expression cassette can be expressed at its 5' and 3' ends, respectively. flanked by 5' ITR and 3' ITR sequences (e.g., any of the 5' or 3' ITR sequences described in Table 12). Generally, AAV2 ITR sequences are contemplated for use in conjunction with the methods and compositions disclosed herein , however, ITR sequences from other AAV serotypes disclosed herein may also be used (see " AAV Vectors " section above). The general architecture of this construct includes at least the following elements oriented in the 5' to 3' direction: (i ) 5' ITR sequence (for AAV vectors only; see Table 12); (ii) promoter sequence (e.g., any of the promoter sequences listed in Table 11); (iii) 5' flanking sequence ( For example, see Table 13); (iv) a stem-loop sequence that is at least 85% identical to any of the following nucleic acid sequences (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: SEQ ID NO: 1 to SEQ ID NO: 19, SEQ ID NO: 34 to SEQ ID NO: 62, SEQ ID NO: 97 to SEQ ID NO: 108, SEQ ID NO: 133 to SEQ ID NO: 147, SEQ ID NO: 226 to SEQ ID NO: 229 and SEQ ID NO : 238 to SEQ ID NO: 241; (v) WPRE sequence, as appropriate; (vi) polyadenylation sequence (e.g., see Table 12); and (vii) 3' ITR sequence (AAV vectors only; see Table 12).

In a specific example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to the following The inhibitory RNA sequence of any one of the SEQ. ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO : 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to An inhibitory RNA sequence having no more than 6 (e.g., no more than 6, 5, 4, 3, 2, or 1) mismatches for any of the following: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO :242 to SEQ ID NO:245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 5 (e.g., no more than 5, 4, 3, 2, or 1) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 4 (e.g., no more than 4, 3, 2, or 1) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 3 (e.g., no more than 3, 2, or 1) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO :245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 2 (e.g., no more than 2 or 1) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79. SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245 . In yet another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 1 mismatch: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245.

In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to An inhibitory RNA sequence that has no more than 10 (eg, no more than 10, 9, 8, 7, or 6) mismatched nucleotides (i.e., mismatches) for any of the following: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 9 (e.g., no more than 9, 8, 7 or 6) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 8 (e.g., no more than 8, 7, or 6) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO :245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 7 (eg, no more than 7 or 6) mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79. SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245 . In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 6 mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 5 mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 4 mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 3 mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 2 mismatches: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245. In another example, a satellite sequence that is substantially complementary to an inhibitory RNA sequence of any of: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233 and SEQ ID NO: 242 to SEQ ID NO: 245) relative to The inhibitory RNA sequence of any of the following has no more than 1 mismatch: SEQ ID NO: 16 to SEQ ID NO: 30, SEQ ID NO: 63 to SEQ ID NO: 79, SEQ ID NO: 109 to SEQ ID NO: 120, SEQ ID NO: 139 to SEQ ID NO: 144, SEQ ID NO: 230 to SEQ ID NO: 233, and SEQ ID NO: 242 to SEQ ID NO: 245.

polygene miRNA box

Flank/trunk-loop/flank constructs (eg, pri-miRNA) can be viewed as single miRNA "cassettes" and can be concatenated (eg, provided in a multigene array driven by one or more promoters). Multiple (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pre-miR backbone-loop sequences can be embedded in any polynucleotide sequence of a longer transcript (e.g., introns) or endogenous microRNA flanking sequences (5' and 3' of each backbone-loop, such as -5p and -3p sequences). Each pre-miR backbone-loop sequence can be expressed under the control of a dedicated promoter (e.g., as a multigene construct with separate promoter sequences, each promoter sequence independently regulating individual pre-miR backbone-loop sequences performance; that is, each promoter operates independently of the other promoter to produce individual microRNAs). It has been shown that flanking sequences that provide at least a 5-bp extension of the backbone are sufficient for backbone-loop processing (Sun et al . BioTechniques. 41:59-63, July 2006, incorporated herein by reference). The spacer sequence may be located between the 3' flanking sequence of the first miRNA expression cassette and the 5' flanking sequence of the second miRNA expression cassette. Spacer sequences may originate from coding or noncoding (e.g., intronic) sequences and be of varying lengths but are not considered part of the stem-loop-flanking sequence (Rousset, F. et al., Molecular Therapy : Nucleic Acids , 14:352-63, 2019, incorporated herein by reference.)

Exemplary expression cassettes can include a nucleotide sequence that includes: (a) a first polynucleotide encoding a first miRNA sequence that includes a guide RNA sequence that hybridizes to Grik2 mRNA; and (b) encoding a second miRNA. The second polynucleotide of the sequence contains the guide RNA sequence that hybridizes to Grik2 mRNA. For example, the expression cassette from 5' to 3' may include: (a) a first 5' flanking region located 5' of the leading strand, said first flanking region including a first 5' flanking sequence identical to its sequence (e.g., see Table 13); (b) first backbone-loop structure, which includes the multi-core of any one of SEQ ID NOs: 1-19, 34-62, 97-108, 133-147, 226-229 and 238-241 The nucleotide (c) is located in the first 3' flanking region 3' of the follower strand (e.g., see Table 13) and the 3' spacer sequence; (d) is located in the second 5' flanking region 5' of the leader strand (e.g., see Table 13) , see Table 13); (e) a second backbone-loop structure, which includes any one of SEQ ID NOs: 1-15, 46-62, 97-108, 133-138, 226-229 and 238-214 The polynucleotide; (f) the second 3' flanking region, including the 3' flanking sequence located 3' of the follower strand (eg, see Table 13). In certain embodiments, the expression cassette from 5' to 3' may include: (a) a first 5' flanking region located at 5' of the leading strand, said first flanking region including a first 5' flanking region that is identical in sequence to Sequence (e.g., see Table 13); (b) a first backbone-loop structure, including any one or more of the polynucleotides of SEQ ID NO: 4, 19, and 34; (c) located 3' of the slave strand and the first 3' flanking region of the 3' spacer sequence (e.g., see Table 13); (d) the second 5' flanking region located 5' of the guide strand (e.g., see Table 13); (e) the second backbone - a loop structure, which includes the polynucleotide of any one or more of SEQ ID NO: 135, 141 and 147; (f) a second 3' flanking region, including a 3' flanking sequence located 3' of the follower strand ( For example, see Table 13). In certain embodiments, the sequences of the first and second backbone-loop structures are reversed such that the first backbone-loop structure includes the polynucleotide of any one of SEQ ID NOs: 135, 141, and 147, and The second backbone-loop structure includes the polynucleotide of any one of SEQ ID NOs: 4, 19, and 34.

The first 5' flanking sequence, the first 3' flanking sequence, the second 5' flanking sequence and the second 3' flanking sequence can be selected from Table 13.

A polycistronic or multigene rAAV expression construct may include a transgene consisting of sequential (eg, contiguous or non-contiguous) miRNA-encoding polynucleotides _X1 , such as ( _X1 ) _n . _The Any of the loop sequences out. The polycistronic transgenic gene has the molecular formula (X ₁ ) _n and is under the control of a single promoter. This single promoter is located at the 5' end of the transgenic gene, so that the molecular formula of the promoter and the transgenic gene is promoter-(X ₁ ) _n , where n is an integer from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Multigene constructs can also include stuffer sequences (eg, SEQ ID NO: 250 or SEQ ID NO: 251) at the 3' end of the construct to increase vector production efficiency.

pair miRNA dual promoter expression cassette

Multigene expression cassettes containing multiple (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pre-miR backbone-loop sequences can include multiple promoter sequences to The performance of each individual pre-miR stem-loop sequence is modulated such that each individual pre-miR stem-loop sequence is operably linked to a dedicated promoter sequence. In this case, _the expression construct has the formula structure (Promoter- _X1 ) _n , where -241, and n is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Additional regulatory elements, such as enhancer sequences, terminator sequences, polyadenylation signal sequences, introns, and/or sequences capable of forming secondary structures, such as any of the regulatory elements disclosed herein, may be operable Groundly connected to the 5' end and/or 3' end of the promoter-X ₁ structure.

In a specific example, a dual-miRNA expression cassette includes two pre-miR backbone-loop sequences, each sequence is under the control of an individual promoter sequence (e.g., the promoter sequences disclosed herein). The two promoters in a dual-miRNA cassette can be the same promoter or different promoters.

In a specific example, the dual-miRNA expression cassette includes the following nucleotide sequences from 5' to 3': (a) a first promoter sequence (such as any promoter sequence disclosed herein, such as Table 11, such as hSyn promoter promoter or CaMKII promoter; (b) a first 5' flanking region located 5' to the first stem-loop sequence (eg, see Table 13); (c) a first stem-loop sequence including SEQ ID NO. : The polynucleotide of any one of 1-19, 34-62, 97-108, 133-147, 226-229 and 238-241; (d) The first nucleotide located 3' of the first guide nucleotide sequence 3' flanking region (e.g., see Table 13); (e) second promoter sequence (e.g., see Table 11), or it has at least 85% (at least 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of the variation, or more (e.g., 100%) identical to its sequence; (f) located in section The second 5' flanking region 5' of the two backbone-loop sequences (for example, see Table 13); (g) the second backbone-loop sequence, including SEQ ID NOs: 1-19, 34-62, 97- The polynucleotide of any one of 108, 133-147, 226-229, and 238-241; (h) a second 3' flanking region located 3' of the polynucleotide (eg, see Table 13). If necessary, hSyn The promoter and the CaMKII promoter can be replaced with a constitutive promoter containing the cytomegalovirus enhancer (eg, CAG or CBA), U6, H1, or 7SK promoter.

In another example, the first promoter is a SYN promoter (e.g., see Table 11) and the second promoter is a CAMKII promoter (e.g., see Table 11).

Can range from 10-1500 (for example, 20-1400, 30-1300, 40-1200, 50-1100, 60-1000, 70-900, 80-800, 90-700, 100-600, 200-500 or 300- The sequence identity of the sequences described in the above dual-miRNA expression box was determined within a range of 400) nucleotides. For example, sequence identity to the dual-miRNA expression cassette described above can be determined over 10 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 20 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 30 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 40 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 50 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 60 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 70 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 80 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 90 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 100 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 150 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 200 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 250 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 300 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 350 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 400 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 450 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 500 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 550 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 600 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 650 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 700 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 750 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 800 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 850 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 900 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 950 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 1000 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 1100 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined over 1200 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 1300 nucleotides. In another example, sequence identity to the dual-miRNA expression cassette described above was determined at 1400 nucleotides. In yet another example, sequence identity to the dual-miRNA expression cassette described above was determined over 1500 nucleotides.

The dual-miRNA expression cassette may include a promoter that is a synaptophysin promoter and/or a calcium/calmodulin-dependent protein kinase II promoter.

MicroRNA loop sequences suitable for use in conjunction with the dual-miRNA expression cassette disclosed herein may be E-miR-30, miR-218-1, or E-miR-124-3 loop sequences.

The dual-miRNA expression cassette of the present disclosure can also incorporate a 5'-ITR at the 5' end of the expression cassette (e.g., see Table 12) and a 3'-ITR at the 3' end of the expression cassette (e.g., see Table 12 ).

Additionally, the dual-miRNA expression constructs disclosed herein may include a first polyadenylation (polyA) signal operably linked between the 3' end of the first 3' flanking region and the 5' end of the second promoter, and or a second polyA signal operably linked between the second 3' end of the second 3' flanking region and the 3' ITR. The first polyA signal and the second polyA signal can be the same (for example, both are RBG or BGH polyA signals) or different (for example, the first polyA signal is RBG polyA, the second polyA signal is BGH polyA; or the first polyA signal is BGH polyA, the second polyA signal is RBG polyA). Improved production of AAV vectors containing multiple miRNA sequences

Preparation of AAV vectors using plasmids encoding single or dual miRNA expression cassettes (e.g., the expression cassettes disclosed herein) may be hampered by improper AAV genome packaging. First, the pri-miRNA sequence is very short (less than 200 bases), which depends on the promoter length, and designing a transgene cassette with a single promoter controlling the expression of a single miRNA may result in an AAV genome that is significantly shorter than that of AAV. Maximum packaging capacity (approximately 4.8 kb). Therefore, a single capsid may be loaded with multiple vector genomes if the expected full genome length is less than 2.4 kb (half the AAV packaging capacity). This can be mediated by polymerase readout without the need for appropriate endonuclease nicking, resulting in AAV genome dimers (or trimers) that can be packaged into AAV if the multimer is of appropriate length. in the shell. This subsequently introduces significant heterogeneity within the AAV vector particle population, which makes the manufacture and characterization of drug products more difficult.

Secondly, due to the inclusion of hairpin structures, transgenic genes based on shRNA and miRNA themselves have significant secondary structures. It has been shown that these internal secondary structures within the AAV genome can act as “pseudo” ITRs during AAV genome replication and packaging, resulting in truncation events and a heterogeneous population of AAV vector particles containing a mixture of complete and partial vector genomes.

We have found that using additional sequences (e.g., additional pre-miRNA backbone-loop sequences, second promoter sequences, filler sequences (e.g., SEQ ID NO: 250 or SEQ ID NO: 251), using self-complementary AAV vectors, etc.) fill AAV genomes with sizes below the AAV packaging capacity, greatly improving AAV packaging by avoiding the incorporation of copy numbers of partial (i.e. truncated) AAV genomes and preventing or reducing multiple packaging events. Therefore, the constructs described herein avoid inappropriate packaging of AAV genomes by incorporating the above sequences into the AAV expression cassette to increase the vector size to a value closer to the maximum AAV packaging capacity. In certain embodiments, the vector contains one or more (e.g., 1, 2, or more) stuffer sequences. In certain embodiments, one or more stuffer sequences are located at the 3' end of the presentation cassette. In certain embodiments, one or more filler sequences are at least 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%) identical to the nucleic acid sequence in SEQ ID NO: 250 % or more (e.g., 100%)) sequence identity. In certain embodiments, one or more filler sequences are at least 90% identical (e.g., at least 91%, 95%, 96%, 97%, 98%, 99%, or more) to the nucleic acid sequence in SEQ ID NO: 250. Multiple (e.g., 100%)) sequence identity. In certain embodiments, one or more filler sequences are at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more (e.g., 100%) identical to the nucleic acid sequence in SEQ ID NO: 250 )) sequence identity. In certain embodiments, one or more filler sequences have at least 99% sequence identity with the nucleic acid sequence in SEQ ID NO:250. In certain embodiments, one or more filler sequences have the nucleic acid sequence in SEQ ID NO:250. In certain embodiments, one or more filler sequences are at least 85% (e.g., at least 86%, 90%, 95%, 96%, 97%, 98%, 99%) identical to the nucleic acid sequence in SEQ ID NO: 251 % or more (e.g., 100%)) sequence identity. In certain embodiments, one or more filler sequences are at least 90% identical to the nucleic acid sequence in SEQ ID NO: 251 (e.g., at least 91%, 95%, 96%, 97%, 98%, 99%, or more Multiple (e.g., 100%)) sequence identity. In certain embodiments, one or more filler sequences are at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more (e.g., 100%) identical to the nucleic acid sequence in SEQ ID NO: 251 )) sequence identity. In certain embodiments, the one or more filler sequences have at least 99% sequence identity with the nucleic acid sequence in SEQ ID NO: 251. In certain embodiments, one or more filler sequences have the nucleic acid sequence in SEQ ID NO: 251.

For example, in some cases, constructs expressing one miRNA under the control of a single promoter or two miRNAs from two different promoters (dual construct approach) may not perform as well as in vitro / ex vivo / in silico evaluations. Perform as predicted in vivo. In these cases, the following strategies can be implemented to establish genome length and thereby generate a homogenous, full-length, individually packaged population of AAV vector particles.

First, if a single miRNA "guide" needs to be expressed, a filler sequence (e.g., SEQ ID NO: 250 or SEQ ID NO: 251) can be added to increase the total length of the AAV vector genome without damaging the promoter or the miR cassette itself. . This stuffer sequence can be added downstream of the transgene cassette (3' to the polyA sequence) . In certain embodiments, the AAV vector comprises at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 95%, 99%) identical to any one of SEQ ID NOs: 252-257 or more (e.g., 100%)) sequence identity. In a preferred embodiment, the AAV vector includes the sequence of any one of SEQ ID NOs: 252-257. The length and content of this padding sequence can be varied while retaining its ability to improve AAV packaging homogeneity. Additionally, this filler sequence can be placed upstream (5') of the promoter. Stuffer sequences can be used with scAAV vectors or single-stranded (ss) AAV vectors.

Secondly, if multiple miRNAs are to be expressed but a single promoter strategy is chosen, vectors can be prepared by concatenating multiple miRNA cassettes. Although using the same scaffold to tandem multiple miRNA cassettes (e.g., up to 5 miRNA cassettes) can lead to recombination between homologous sequences within the vector gene, using different scaffolds to tandem miRNA cassettes with non-homologous flanking and looping sequences can improve this. Packaging issues. If inclusion of additional miRNA cassettes does not result in vector gene bodies of appropriate length, filler sequences can be incorporated as described above to increase length to increase packaging efficiency. In certain embodiments, the AAV vector comprises at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 95%, 99%) identical to any one of SEQ ID NOs: 252-257 or more (e.g., 100%)) sequence identity. In a preferred embodiment, the AAV vector includes the sequence of any one of SEQ ID NOs: 252-257.

insect expression vector

The inhibitory polynucleotides described herein may also be encoded in a suitable insect expression vector (e.g., a viral vector, such as a baculovirus viral vector) and expressed in an insect expression system. In certain embodiments, inhibitory polynucleotides described herein can be incorporated into a nucleic acid vector (e.g., a baculovirus or baculovirus-based vector, a retroviral vector, or other viral vector), transfected to an insect cell line (e.g., Sf9 cells) and cultured under conditions that allow expression of the polynucleotide. For example, insect cell lines (e.g., Sf9 cells) can be transiently or stably transfected using inhibitory polynucleotides of the present disclosure.

Baculovirus delivery vectors and their corresponding modified host cells are commercially available, such as pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, pAcAB4; pBAC-3, pBAC6, pBACgus-6, pBACsurf-1, pPolh-FLAG and pPolh-MAT. Methods for baculovirus and insect cell expression systems are well known in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) and Luckow and Summers, Bio/Technology 6:47 (1988) are incorporated herein by reference. Those skilled in the art will understand that the expression system is not limited to the baculovirus expression system. Importantly, the expression system allows expression of inhibitory polynucleotides of the present disclosure. For example, other suitable insect expression systems include the entomopoxvirus system (insect mother virus) and the cytoplasmic polyhedrosis virus (CPV) system.

bacterial expression vector

Standard bacterial vectors include, for example, phages X and M13, and plasmids such as pBR322, pSKF and pET23D. In certain embodiments, an inhibitory polynucleotide described herein, or a nucleic acid vector encoding the same, can be incorporated into a plasmid or alternative bacterial expression vector and cultured under conditions that allow expression of the polynucleotide. Cloning and expression vectors suitable for use in bacterial host cells are described by Pouwels et al. (Selective vectors: a laboratory manual, Elsevier, New York, 1985). Exemplary bacterial strains may include E. coli , Bacillus subtilis , Salmonella enterica , or any bacterial strain capable of expressing a heterologous polynucleotide. Elements commonly included in bacterial expression vectors include a replicon (a gene encoding antibiotic resistance that allows selection of bacteria containing recombinant plastids), and unique restriction sites in non-essential regions of the plastid that allow the insertion of recombinant sequences. pharmaceutical composition

The inhibitory polynucleotides described herein, or nucleic acid vectors encoding the same, can be formulated into pharmaceutical compositions for administration to mammalian (eg, human) subjects in a biocompatible form suitable for in vivo administration.

The compositions disclosed herein may be formulated in any suitable carrier for delivery to a subject (e.g., human). For example, a pharmaceutically acceptable suspension, dispersion, solution or emulsion may be formulated. Suitable media include saline and liposomal formulations. More specifically, pharmaceutically acceptable carriers may include non-aqueous sterile aqueous solutions, suspensions, and emulsions. Recombinant human albumin (rAlbumin Human NF RECOMBUMIN® Prime) can also be used as a stabilizer for AAV vectors (Albumedix, Nottingham, UK). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Carriers suitable for intravenous administration include liquid and nutritional supplements, electrolyte supplements (such as supplements based on Ringer's Dextrose Injection), etc.

Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases may also be present.

Colloidal dispersion systems can also be used for targeted gene delivery. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, microcells, mixed microcells, and liposomes.

The compositions described herein can be used in the free base form, in salt form, solvates and as prodrugs. All forms are within the methods described in this article. According to the methods of the present disclosure, the described compounds or salts, solvates or prodrugs can be administered to a patient in a variety of forms depending on the chosen route of administration.

Thus, the compositions described herein may be formulated for administration, for example, by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, intranasal, rectal, patch, pump, or dermal penetration, and formulated accordingly Pharmaceutical compositions. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal and topical administration. Parenteral administration can be by continuous infusion over a selected period of time.

Solutions of the agents described herein can be prepared in water moderately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerin, liquid polyethylene glycol, DMSO, and their mixtures and oils with or without alcohols. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms. General procedures and ingredients for selecting and preparing appropriate formulations are described in, e.g., Remington's Pharmaceutical Sciences (2012, 22nd ed.) and United States Pharmacopeia: National Formulary (USP 41 NF 36), published in 2018. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be free-flowing and ready for administration via a syringe. Local, regional or systemic administration may also be appropriate. The compositions described herein can be advantageously contacted by administering an injection or multiple injections to the target site, for example, spaced approximately 1 centimeter apart.

The compositions described herein may be administered to animals (e.g., humans) alone or in combination with a pharmaceutically acceptable carrier, in proportions that depend on the solubility and chemical properties of the compounds, the chosen route of administration, and standard medical practice, as described herein. norm.

Accordingly, the present disclosure provides a pharmaceutical composition comprising an inhibitory RNA agent (eg, siRNA, shRNA, miRNA or shmiRNA) disclosed herein. In particular, the present disclosure provides compositions comprising a vector comprising an inhibitory RNA agent of the present disclosure. In specific examples, the present disclosure provides pharmaceutical compositions comprising a vector (eg, a lentiviral or AAV vector) that includes an inhibitory RNA of the present disclosure operably linked to a promoter, as disclosed herein. The pharmaceutical composition may comprise an AAV vector comprising (a) a viral capsid; and (b) an artificial polynucleotide comprising a presentation cassette flanked by an AAV ITR, wherein the presentation cassette comprises a polynucleotide encoding an inhibitory polynucleotide , the inhibitory polynucleotide binds to and inhibits the expression of Grik2 mRNA and is operably linked to one or more regulatory sequences that control the expression of the polynucleotide in CNS cells.

The inhibitory RNA agents disclosed herein can be combined with pharmaceutically acceptable excipients and optional sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to mammals, especially humans (where appropriate). A pharmaceutically acceptable carrier or excipient means any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. The pharmaceutical compositions disclosed herein may be formulated for intracerebral (e.g., intraparenchymal or intracerebroventricular), intramuscular, intravenous, skin-penetrating, topical, oral, sublingual, subcutaneous, or rectal administration.

The active ingredient of the composition (eg, an inhibitory RNA agent targeting Grik2 ), alone or in combination with another therapeutic agent, can be administered to a subject in need thereof as a mixture with conventional pharmaceutical supports in a unit dosage form. Suitable unit dosage forms include oral route forms (such as tablets, gel capsules, powders, granules and oral suspensions or solutions), sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical , intraperitoneal, intramuscular, intravenous, subcutaneous, skin penetration, intrathecal, intracerebral, stereotaxic and intranasal administration forms as well as rectal administration forms. Typically, for injectable formulations, the pharmaceutical composition includes a pharmaceutically acceptable carrier. These can be in particular isotonic, sterile, saline solutions (mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, in particular The freeze-dried composition, which at the time of addition depends on whether it is sterile water or physiological saline, can be formulated as an injection solution.

Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil, or aqueous propylene glycol solutions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be free-flowing to facilitate injection. It must remain stable under the conditions of manufacture and storage and must be resistant to the contaminating effects of microorganisms such as bacteria and fungi. Solutions including compounds of the present disclosure as free bases or pharmaceutically acceptable salts can be prepared in water moderately mixed with a surfactant such as hydroxypropylcellulose.

Dispersions can also be prepared in glycerin, liquid polyethylene glycols, mixtures thereof and oils. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms. The inhibitory polynucleotide agents disclosed herein can be formulated as neutral or salt form compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amine groups of proteins), and salts with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.) class. Salts formed with free carboxyl groups can also be derived from: inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine , procaine, etc. The carrier can also be a solvent or dispersion medium, such as water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), appropriate mixtures thereof, and vegetable oils. Proper flowability can be maintained by using coatings (eg lecithin), by maintaining the required particle size (for dispersions) and by using surfactants.

The action of microorganisms can be prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In some cases, pharmaceutical compositions of the present disclosure may include an isotonic agent, such as sugar or sodium chloride.

Prolonged absorption of the injectable compositions may be prolonged by the use of agents that delay absorption such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the required amount of the active agent in the appropriate solvent and various other ingredients enumerated below as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients as disclosed herein. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods may be vacuum drying and freeze-drying techniques, which can obtain a powder of the active ingredient and any other desired ingredients from a previously filtered sterile solution. Once formulated, the solution will be administered in a manner compatible with dosage requirements and in a therapeutically effective amount.

These formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions mentioned above, but drug release capsules may also be used. For example, for parenteral administration in aqueous solutions, the solution should be appropriately buffered if necessary and the liquid diluent first made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Sterile aqueous media that can be used are well known in the art. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection or injected at a designated infusion site. Certain variations in dosage will necessarily occur depending on the subject being treated. In any case, the administering practitioner can use appropriate patient information and art-recognized methods to determine the appropriate dosage for the individual subject. diagnostic methods

A subject (e.g., a human subject) can be diagnosed with epilepsy (e.g., TLE), for example, using methods well known in the art, and thus identified as being in need of treatment using the compositions and methods disclosed herein. For example, the diagnosis of epilepsy in a subject can be guided by neurophysiological testing to identify epileptogenic lesions in the subject's brain and the severity of epileptiform activity. Example neurophysiological testing methods well known in the art include electroencephalography (EEG), magnetoencephalography (MEG), functional MRI (fMRI), single photon emission computed tomography (SPECT), and positron emission tomography (PET) . EEG and MEG provide continuous measurements of cortical function with high temporal resolution and aid in the detection of interictal (the period between epileptic seizures) epileptiform discharges, which may indicate changes in the subject's epilepsy disorder. Positive diagnosis. The subject's brain activity can be compared to standards appropriate for the subject's age, medical history, and lifestyle (e.g., a reference population, such as a non-epileptic patient population) to determine a diagnosis of epilepsy in the subject.

Subjects may be diagnosed with any of a variety of epilepsy conditions, including but not limited to TLE (e.g., mTLE or lTLE), benign Rolandic epilepsy, frontal lobe epilepsy, infantile nodding spasms, juvenile myoclonic epilepsy, adolescent Absence epilepsy, childhood absence epilepsy (pyknolepsy), hot water epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravet syndrome, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen syndrome, borderline epilepsy, epilepsy Retention status, abdominal epilepsy, massive bilateral myoclonus, menstrual epilepsy, Jacksonian epilepsy, Lafora syndrome, and photosensitive epilepsy. In cases where the epilepsy condition is TLE, TLE may be characterized by focal or generalized seizures.

Based on the revealed method, the epileptogenic lesions can be localized to specific brain regions (such as the medial temporal lobe, lateral frontal lobe, frontal lobe, etc.) and the patient's epilepsy type can be diagnosed. Electrophysiological characterization of epileptic brain activity can also be used to identify specific types or subtypes of epilepsy in subjects. For example, the presence of fast (250-600 Hz) spike-wave ripples (SPW-Rs) in cortical regions such as the hippocampus or cerebral cortex may indicate a positive diagnosis of TLE in a subject. In another example, Lennox-Gastaut syndrome is often characterized by fast electrographic oscillations (10-15 Hz) recorded in the neocortex and thalamus. In addition, if the patient exhibits obvious behavioral manifestations of an epileptic seizure, such as generalized convulsions, temporary absence of consciousness (decreased level of consciousness lasting approximately 10 seconds), nervousness, myoclonus, bowel or bladder loss, tongue biting, fatigue, headache, difficulty speaking, , abnormal behavior (e.g., immobile gaze or spontaneous movements of the hands or mouth), psychosis, and/or focal weakness, imaging monitoring of subjects in the inpatient setting may indicate a diagnosis of epilepsy in the patient. Self-reported symptoms in subjects diagnosed with epilepsy may also indicate a positive diagnosis. Such self-reported symptoms may include familiar or never-before-seen sensations, auras, amnesia, spontaneous and unprovoked fear and anxiety, nausea, auditory, visual, olfactory, gustatory or tactile hallucinations, visual distortions (e.g. visual distortions) major or micropsia), dissociation or derealization, joint sensations, irritability or euphoria, fear, anger, or indescribable feelings. medicinal purposes

Disclosed herein are methods of treating epilepsy (eg, TLE) in subjects diagnosed with or at risk of developing the epilepsy disorder by administering the above compositions (eg, inhibitory RNA agents or nucleic acid vectors encoding the same). After administration, the inhibitory RNA reagents of the present disclosure are able to bind to Grik2 mRNA and inhibit its expression. Targeting of Grik2 by inhibitory RNA reagents disclosed herein can demonstrate that a first cell or group of cells (e.g., neural cells; such cells may be present, e.g., in a subject or in a sample derived from the subject Medium) exhibits a decrease in Grik2 mRNA concentration in which the cell transcribes Grik2 and the cell has been treated or has been treated (e.g., by contacting one or more cells with an inhibitory polynucleotide of the disclosure, or by administering an inhibitory polynucleotide of the disclosure) polynucleotide to a subject in which the cell exists or has existed). In a specific example, the expression of Grik2 is reduced in a first cell or group of cells compared to a second cell or group of cells that is substantially the same as the first cell or group of cells but is not or has not been After such treatment (control cells are not treated with inhibitory polynucleotides or are not treated with inhibitory polynucleotides that target the gene of interest). The degree of reduction in the mRNA concentration of the gene of interest (eg Grik2 ) can be expressed in the following way:

Changes in the expression concentration of a gene (eg, the Grik2 gene) can be assessed based on a decrease in a parameter related to the expressed function of the gene of interest, such as protein expression of the gene of interest or signaling downstream of the protein. Changes in the expression concentration of a gene of interest can be determined in any cell expressing the gene of interest, whether endogenous or heterologous from the expression construct, and by any assay known in the art.

Changes in Grik2 expressed concentration can be evidenced by a decrease in GluK2 protein concentration expressed by a cell or group of cells (e.g., derived from the GluK2 protein concentration expressed in a subject's sample). As described above, to assess Grik2 mRNA inhibition, changes in the apparent concentration of GluK2 protein in treated cells or groups of cells can similarly be expressed as a percentage of the protein concentration in control cells or groups of cells.

Control cells or cell groups that can be used to evaluate changes in Grik2 gene expression include cells or cell groups that have not been contacted with inhibitory polynucleotides of the present disclosure. For example, control cells or groups of cells may be derived from an individual subject (eg, a human or animal subject) prior to treating the subject with an inhibitory polynucleotide.

The Grik2 mRNA concentration expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. For example, the apparent concentration of Grik2 mRNA in a sample can be determined by detecting the transcribed polynucleotide or portion thereof (eg, mRNA). RNA can be extracted from cells using RNA extraction techniques, including, for example, the use of acid phenol/guanidinium isothiocyanate extraction (RNAzol B; Biogenesis), RNEASY ^™ RNA Preparation Kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA can be detected using the method described in PCT publication WO2012/177906, the entire content of which is incorporated herein by reference. Nucleic acid probes can also be used to determine the expressed concentration of a gene of interest. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific sequence (eg, mRNA). Probes may be synthesized or derived from appropriate biological agents using methods well known and routine in the art. Probes can be specially designed for labeling. Examples of molecules that can serve as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. A method for determining mRNA concentration that involves contacting isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA of the gene of interest. The mRNA can be immobilized on a solid surface and contacted with the probe, such as by performing separation of the mRNA on an agar gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. Probes can also be immobilized on a solid surface and the mRNA contacted with the probe, such as in the AFFYMETRIX GeneChip Array. mRNA detection methods known in the art can be adapted to determine the mRNA concentration of the gene of interest.

Alternative methods for determining the expression concentration of a gene of interest in a sample involve amplification of nucleic acids such as mRNA in the sample and/or reverse transcriptase (for preparation of cDNA) processes, such as by RT-PCR (Experimental example by Mullis, 1987 , U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc.Natl.Acad.Sci. United States 88:189-193), self-sustaining sequence replication (Guatelli et al. (1990) Proc.Natl.Acad.Sci .USA 87:1874-1878), transcription amplification system (Kwoh et al. (1989) Proc.Natl.Acad.Sci. USA 86:1173-1177), Q-Beta replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, and the amplified molecules are then detected using techniques well known in the art. These detection protocols are particularly useful for detecting nucleic acid molecules if the number of such nucleic acid molecules is very small. In specific aspects of the present disclosure, the expression concentration of the gene of interest is determined by quantitative fluorescent RT-PCR (i.e., TAQMAN ^™ System) or DUAL-GLO® Luciferase Assay.

Genes of interest can be monitored using membrane blots (such as for hybridization assays such as Northern, Southern, blot, etc.) or microwells, sample tubes, gels, beads or fibers (or any solid support, including bound nucleic acids) Expression of mRNA concentration. See U.S. Patent Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. Determination of gene expression concentrations may also involve the use of nucleic acid probes in solution.

Expressed mRNA concentrations can also be assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of this PCR method is described and exemplified in the examples provided in this article. Such methods can also be used to detect nucleic acids for genes of interest.

Furthermore, the concentration of GluK2 protein produced by expression of the Grik2 gene can be determined using any method known in the art for measuring protein concentration. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, colorimetric assays , Spectrophotometry, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-binding immunosorbent assay (ELISA), immunofluorescence assay method, electrochemiluminescence assay, etc. Such assays can also be used to detect proteins indicating the presence or duplication of proteins produced by the gene of interest. In addition, the above-described assays can be used to report changes in the mRNA sequence of interest that result in restoration or alteration of protein function, thereby providing therapeutic effects and benefits to the subject, treating the subject's condition, and/or reducing the symptoms of the subject's disease. .

Accordingly, the above-described assays for measuring Grik2 mRNA or GluK2 protein expression can be used to identify subjects in need of therapeutic treatment (e.g., subjects with epilepsy, such as TLE) using one or more inhibitory RNAs disclosed herein reagent (eg, any of the inhibitory RNA reagents described in Table 2) or a nucleic acid vector encoding the same treatment. For example, a patient identified as having TLE may exhibit epileptogenic lesions within the temporal lobes of one hemisphere of the brain, resulting in uncontrollable (eg, treatment-resistant, eg, chronic) seizures. As discussed herein, such epileptogenic lesions may be caused by, for example, abnormal growth of recurrent dentate granule cell mossy fibers and aberrant (i.e., increased) expression of Grik2 at recurrent synapses formed by such mossy fibers. . Using the assays described above, it can be determined whether a subject would benefit from treatment with one or more Grik2 inhibitory RNA agents disclosed herein, for example, by examining brain tissue collected from the hippocampus of the epileptogenic hemisphere and the same region of the healthy hemisphere. Perform small biopsies. Demonstrate that the epileptogenic hemisphere exhibits higher concentrations (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%) than the unaffected hemisphere. ,50%,55%,60%,65%,70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98 %, 99% or higher (e.g., 100%)) Grik2 mRNA or GluK2 protein expression concentrations would indicate that the patient may benefit from treatment using the methods and compositions disclosed herein. If a subject with TLE develops epileptogenic lesions in both cerebral hemispheres, the analysis described above can be used to compare hippocampal tissue obtained from one or more hemispheres of a subject affected by TLE with that of healthy controls. Grik2 mRNA or GluK2 protein concentrations between hippocampal tissues obtained from the same hemisphere of the subject (eg, postmortem tissue from subjects without TLE). Show that the epileptogenic hemisphere of subjects affected by TLE exhibits higher (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more (e.g., 100%)) Grik2 mRNA or GluK2 protein expression concentrations, would indicate that subjects affected by TLE would benefit from use of the compositions of the present disclosure and methods of treatment. Grik2 mRNA concentrations or GluK2 protein concentrations in neuronal cells of subjects suspected of needing treatment may also be compared to standard or reference concentrations of these analytes known to be indicative of disease states.

Additionally, the assays described above can be used to determine whether a subject (eg, suffering from an epilepsy disorder, such as TLE) responds to treatment using the compositions and methods disclosed herein. For example, as described above, hippocampal brain tissue from the epileptogenic cerebral hemisphere can be obtained via small biopsies from subjects affected by TLE prior to treatment with the compositions and methods disclosed herein, and the assays described above can be used. Assess the performance of Grik2 mRNA or GluK2 protein. The subject can then be administered medication and treatment according to the methods and compositions disclosed herein. After the patient recovers from treatment with the methods and compositions of the present disclosure (e.g., 1, 5, 10, 15, 30, 60, 90, or more days after treatment), a second test can be performed on the same brain region assessed prior to treatment. Subbiopsies are taken and the concentration of Grik2 mRNA or GluK2 protein can be assessed again. Subjects shown to be affected by TLE exhibit lower (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g. 100%)) The expressed concentration of Grik2 mRNA or GluK2 protein will indicate that the subject is responsive to treatment. Alternatively, Grik2 mRNA or GluK2 protein concentrations can be compared to the same performance from one or more healthy control subjects. Showing that Grik2 mRNA or GluK2 protein concentrations in a TLE-affected patient after treatment are statistically indistinguishable from those in one or more healthy control subjects would indicate that the patient is responding to treatment. Grik2 mRNA concentrations or GluK2 protein concentrations in the neural cells of treated subjects can also be compared to standard or reference concentrations of these analytes, which are known to be indicative of the absence of disease states. Treatment

Subjects suffering from epilepsy (e.g., TLE) can be treated using the compositions and methods described herein. The composition (e.g., a composition containing an inhibitory RNA agent or containing the same carrier) can be administered as a prophylactic treatment to a subject in need thereof (e.g., a subject diagnosed with or at risk for epilepsy (e.g., TLE). Subjects at risk of developing epilepsy may exhibit early symptoms of epilepsy or may not yet have symptoms at the time of treatment.

Route of administration

The compositions disclosed herein may be administered to a subject (e.g., a subject identified as having TLE) using standard methods. For example, the compositions disclosed herein may be administered by any of a variety of different routes, including, for example, systemic administration. Non-limiting examples of systemic administration include enteral (e.g., oral) or parenteral (e.g., intravenous, intraarterial, transmucosal, intraperitoneal, epidermal, intramucosal (e.g., intranasal or sublingual), intramuscular, or transcutaneous administration .Other routes of administration may include intradermal, subcutaneous and transdermal injection.

The compositions disclosed herein may also be administered using methods suitable for local delivery of inhibitory RNA agents or nucleic acid vectors encoding the same. Non-limiting examples of topical administration include epidermal (e.g., topical), intra-articular, and inhalation routes. In particular, the compositions of the present disclosure can be administered topically to the brain tissue (e.g., nerve cells, such as neurons and/or stellate cells) of a subject.

In particular, inhibitory RNA agents and nucleic acid vectors encoding the same can be administered locally to brain tissue in a subject, such as brain tissue determined to exhibit increased epileptiform activity. Local administration to the brain generally involves any method suitable for delivering an inhibitory RNA agent or a nucleic acid vector encoding the same to brain cells (e.g., nerve cells) such that at least a portion of the cells and components of a selected, synaptically connected cell population get in touch with. Vectors can be delivered to any cell in the CNS, including neurons, stellate cells, or both. Typically, vectors are delivered to cells of the CNS, including, for example, the spinal cord, brainstem (medulla, pons, and midbrain), cerebellum, diencephalon (eg, thalamus and hypothalamus), telencephalon (eg, striatum, cerebral cortex (eg, striatum) cortical areas in the occipital, temporal, parietal, or frontal lobes) or combinations thereof, or any suitable cell subpopulation therein. Further transmission sites include the red nucleus, amygdala, entorhinal cortex, and ventrolateral thalamus Neurons in the nucleus or anterior nucleus.

The vectors of the present disclosure can be delivered directly into the parenchyma of the CNS or the brain ventricles via stereotaxic injection or microinjection. In specific examples, vectors of the present disclosure can be delivered directly to one or more epileptic lesions in the subject's brain. For example, vectors of the present disclosure may be administered to a subject via stereotactic injection directly into one or both hemispheres of the heterologous cortex (e.g., hippocampus) or neocortex (e.g., frontal lobes). In specific examples, a vector of the present disclosure is administered to a subject via stereotaxic injection directly into one or both hemispheres of the hippocampus. Alternatively, vectors of the present disclosure may be administered via intravenous injection, such as in the case of vectors that exhibit tropism for CNS tissue, including but not limited to AAV5, AAV9, or AAVrh10.

To specifically deliver the vectors of the present disclosure to specific regions and specific CNS cell populations, the vectors can be administered via stereotaxic microinjection. For example, the subject may have a stereotaxic frame base (screwed into the skull) that is surgically fixed in place. Use high-resolution MRI to image the brain with a stereotaxic frame base (e.g., an MRI-compatible stereotaxic frame base with fiducial markers). The MRI images are then transferred to a computer running stereotaxic software. A series of coronal, sagittal and axial images are used to determine the target injection site and trajectory of a cannula or injection needle used to inject compositions of the present disclosure into the brain. The software directly converts trajectories into three-dimensional coordinates suitable for the stereotaxic frame. A stereotaxic device is placed by drilling a hole above the access site and inserting the injection needle to a specific depth. Compositions, such as those disclosed herein, can be injected into the target site. In cases where the composition includes an integrated vector rather than the production of viral particles, the spread of the vector is minor and is primarily a function of passive diffusion from the injection site. The degree of diffusion can be controlled by adjusting the ratio of carrier to fluid carrier.

Additional routes of administration may also include the application of localized vectors under direct visualization, such as superficial cortical applications or other non-stereotaxic applications. The vector can be delivered intrathecally (e.g., directly into the cisterna magna), intracerebroventricularly (e.g., using intracerebroventricular (ICV) injection), or by intravenous injection.

In one example, the methods of the present disclosure include intracerebral or intracerebroventricular administration via stereotaxic injection. However, other known delivery methods may also be modified in accordance with the present disclosure. For example, in order to distribute a composition more widely throughout the CNS, it can be injected into the cerebrospinal fluid, for example via lumbar puncture. To direct the composition to the peripheral nervous system, it can be injected into the spinal cord, one or more peripheral ganglia, or subcutaneously (subcutaneously or intramuscularly) at the body site of interest. In some cases, the compositions may be administered via the intravascular route. For example, the compositions can be administered intraarterially (carotid artery) with or without disruption of the blood-brain barrier. In addition, for more comprehensive delivery, the composition can be administered during the "opening" of the blood-brain barrier by infusion of a hypertonic solution including mannitol.

The most appropriate route of administration in any particular case will depend on the specific composition of the drug administered, the subject, the specific epilepsy being treated, the method of pharmaceutical formulation, the method of administration (e.g., time and route of administration), the subject's Age, weight, sex, severity of disease being treated, subject's diet, and subject's excretion rate.

combination therapy

The compositions disclosed herein can be administered to a subject in need thereof (e.g., a human subject) in combination with one or more additional treatment modalities (e.g., 1, 2, 3 or more other treatment modalities). Epilepsy, including other therapeutic agents or physical intervention (e.g., rehabilitation therapy or surgical intervention). Two or more agents can be administered simultaneously (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes, or less). These agents can also be administered simultaneously through combined formulations. Two or more agents may also be administered sequentially, such that the effects of the two or more agents overlap and the combined effect results in relief of symptoms, or other parameters associated with the disease, that is greater than either agent or treatment alone or in the absence of Parameters observed with another agent or treatment. The effects of two or more treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Administration of each therapeutic agent may be performed sequentially or substantially simultaneously by any suitable route, including, but not limited to, oral, intravenous, intramuscular, topical, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination can be administered by intravenous injection, while a second therapeutic agent of the combination can be administered topically in a microcartridge into which the compounds are infiltrated. Up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours before or after the second therapeutic agent hours, up to 12 hours, up to 13 hours, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours to 24 hours, or up to 1 -7, 1-14, 1-21 or 1-30 days before the first therapeutic agent can be administered immediately.

In cases where the subject is diagnosed with epilepsy or is at risk of developing epilepsy (e.g., TLE), the second therapeutic agent may include one or more antiepileptic drugs (AEDs), including but not limited to valproic acid, lamotrigine , ethosuximide, topiramate, lacosamide, levetiracetam, clobazam, stiripentol, benzodiazepines, phenytoin, carbamazepine, pyrimidinedione, phenobarbital, Gabapentin, pregabalin, tiagabine, zonisamide, felbamate and/or vigabatrin. In some cases, the second treatment modality may be surgical intervention, such as removal of the epileptogenic brain region (eg, temporal lobectomy) using methods well known in the art, such as radiosurgery (eg, gamma knife or laser ablation). Other treatment modalities that may be administered with the methods and compositions of the present disclosure include vagus nerve stimulation, deep brain stimulation, transcranial magnetic stimulation, and ketogenic diet.

In specific examples, the subject may be administered an immunosuppressive agent, including a regimen of corticosteroids alone or tacrolimus or rapamycin (sirolimus), for example, in combination with mycophenolic acid or with a corticosteroid such as adrenaline. Corticosterone and/or methylpenic cortisol combination. Other immunosuppressive regimens well known in the art may be used in conjunction with the methods and compositions of the present disclosure. Such immunosuppressive treatments may be administered before, after, or concurrently with administration of inhibitory nucleic acid molecules (e.g., inhibitor RNA agents) described herein.

Give medication

Subjects who can be treated as described herein are subjects diagnosed with or at risk of developing epilepsy (e.g., TLE). Subjects who may be treated using the methods and compositions of the present disclosure include, for example, subjects who have previously received one or more therapeutic interventions related to the treatment of epilepsy or subjects who have not previously received a therapeutic intervention for the treatment of epilepsy.

The inhibitory RNA agent of the present disclosure can be administered in an amount and at a time effective to cause one or more (e.g., 2 or more, 3 or more, 4 or more) of the following: (a) reducing Grik2 mRNA in the cells of the subject and/or concentration of GluK2 protein, (b) delay in disease onset, (c) increased subject survival, (d) increased progression of subject free survival, (e) restoration or alteration of GluK2 protein function, ( f) reduce the risk of seizure recurrence; (g) reduce CNS excitotoxicity and associated neuronal cell death; (h) restore physiological excitatory-inhibitory balance in affected areas of the CNS (e.g., hippocampus); and/or (i) Reduce one or more symptoms of epilepsy (e.g., frequency, duration, or intensity of seizures, weakness, absence, sudden confusion, difficulty understanding or producing speech, impaired cognition, difficulty moving, dizziness or loss of balance or coordination, paralysis, and Emotional disorders).

Accordingly, the present disclosure relates to a method of treating epilepsy (e.g., TLE) in a subject in need thereof, wherein the method includes administering an effective amount of a vector comprising an inhibitory RNA (e.g., ASO, such as siRNA, shRNA , miRNA or shmiRNA) inhibitory polynucleotide, this polynucleotide specifically binds to Grik2 mRNA and inhibits the expression of GluK2 protein in the subject. In particular, the present invention provides a method of treating epilepsy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitory RNA agent disclosed herein or a nucleic acid vector encoding the same.

Epilepsy treated using the disclosed methods and compositions may be TLE (e.g., mTLE or lTLE), benign Rolandic epilepsy, frontal lobe epilepsy, infantile nodding spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, and childhood absence epilepsy (pyknolepsy), hot water epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravet syndrome, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen syndrome, borderline epilepsy, epileptic status epilepticus, abdominal epilepsy, Massive bilateral myoclonus, catamenial epilepsy, Jacksonian epilepsy, Lafora syndrome, and photosensitive epilepsy. For example, a subject may be diagnosed with TLE (eg, mTLE or lTLE), such as TLE characterized by focal or generalized seizures. In some cases, epilepsy can be chronic, such as refractory epilepsy (i.e., drug-resistant epilepsy, such as drug-resistant TLE).

As discussed herein, to treat epilepsy and ameliorate the symptoms of epileptic seizures and epileptiform discharges, useful polynucleotides can be deployed through vectors encoding functional RNA (e.g., siRNA, shRNA, miRNA, or shmiRNA) that inhibit the expression of Grik2 mRNA Performance.

The compositions of the present disclosure may be administered in an amount determined to be appropriate by one skilled in the art. In some cases, rAAV was expressed at 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ genome copy numbers per subject ( GC) dose administration. In certain embodiments, rAAV is expressed at 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ GC per kilogram (total weight of the subject) Dosage administration.

In some cases, dose 1 x 10 ¹² to 5 x 10 ¹⁴ GC. In some cases, a fixed dose of 1 x 10 ¹² to 5 x 10 ¹⁴ GC is administered to pediatric or adult patients.

In some cases, the dose is measured by the number of GCs administered per gram of the patient's brain mass into the patient's cerebrospinal fluid (CSF) (eg, intrathecal injection, eg, via suboccipital puncture or lumbar puncture). In some cases, 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ genome copies per gram of patient brain mass Quantity dose. In some cases, the dose was administered at 1 × 10 ⁵ genome copies per gram of patient brain mass. In some cases, the dose was administered at 1 × 10 ⁶ genome copies per gram of patient brain mass. In some cases, the dose was administered at 1 × 10 ⁷ genome copies per gram of patient brain mass. In some cases, the dose was administered at 1 × ¹⁰⁸ genome copies per gram of patient brain mass. In some cases, the dose was administered at 1 × ¹⁰⁹ genome copies per gram of patient brain mass. In some cases, doses were administered at 1 × 10 ¹⁰ genome copies per gram of patient brain mass. In some cases, doses were administered at 5 × 10 ¹⁰ genome copies per gram of patient brain mass. In some cases, doses are administered at 1 x ¹⁰⁹ to 1 x ¹⁰¹¹ genome copies per gram of patient brain mass. In some cases, doses are administered at 1 x 10 ⁹ to 5 x 10 ¹⁰ genome copies per gram of patient brain mass. In some cases, doses were administered at 2 x 10 ⁹ to 9 x 10 ¹⁰ genome copies per gram of patient brain mass. In some cases, doses were administered at 5 x ¹⁰⁹ to 1 x ¹⁰¹¹ gene copy number per gram of patient brain mass. In other cases, doses are administered at 1 x 10 ¹⁰ to 5 x 10 ¹⁰ genome copies per gram of patient brain mass. In other embodiments, the dose is administered at a genome copy number of 1 x 10 ¹⁰ to 9 x 10 ¹⁰ per gram of patient brain mass. An estimate of the patient's (subject's) brain weight is obtained from the MRI brain volume measurement, which is converted to brain mass and used to calculate the precise dosage. It is also possible to use published databases to estimate brain weight based on age ranges.

Optionally, the agents of the present disclosure may be administered as part of a pharmaceutically acceptable composition suitable for delivery to a subject, as described herein. The agents of the present disclosure are included in these compositions in amounts sufficient to provide the desired dosage and/or cause a therapeutic benefit, as can be readily determined by one skilled in the art.

The disclosed compositions described herein can be administered in an amount (eg, an effective amount) and for a time sufficient to treat a subject or achieve one of the results described above (eg, alleviate one or more disease symptoms in the subject). The compositions of the present disclosure may be administered in one or more times. The disclosed composition can be administered once a day, twice a day, three times a day, once every two days, once a week, twice a week, three times a week, once every two weeks, or monthly. Once, every two months, twice a year, or once a year. Treatment may be discontinuous (eg, injection) or continuous (eg, treatment via implant or infusion pump). Depending on the composition used in the treatment and the route of administration, it can be 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or Assess treatment efficacy in subjects over a longer period of time. This article discloses methods for assessing the efficacy of treatments (see, for example, "Pharmaceutical Uses"). Depending on the results of the evaluation, treatment may be continued or discontinued, the frequency or dosage of treatment may be changed, or the patient may be treated with a different disclosed composition. Subjects may receive treatment, or treatment, for a discrete period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) or until remission of the disease or condition is achieved May be chronic, depending on the severity and nature of the disease or condition being treated. For example, a subject diagnosed with TLE and treated with a composition disclosed herein, if initial or subsequent rounds of treatment do not produce a therapeutic benefit (e.g., a reduction in any of the symptoms disclosed herein or Grik2 mRNA or the subject's affected decrease in GluK2 protein concentration in the brain region), one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional treatments may be administered. set

The present disclosure also provides a kit comprising a composition disclosed herein that inhibits expression of the Grik2 gene in a subject (e.g., inhibitory RNA targeting Grik2 mRNA) for preventing or treating epilepsy (e.g., TLE, such as Treatment-refractory TLE). The kit may optionally include an agent or device for delivering the composition to a subject. In other examples, the kit may include one or more sterile applicators, such as syringes or needles. In addition, the kit may optionally include other agents, such as anesthetics or antibiotics. The kit may also include instructions for a user of the kit (such as a physician) to perform the methods disclosed herein. Example

The following examples are presented to provide those of ordinary skill in the art with a description of how to use, prepare, and evaluate the compositions and methods described herein, and are intended to be examples of the present disclosure only and are not intended to limit what the inventors believe to be theirs. Scope of disclosure. Example 1 : Design and synthesis of inhibitory polynucleotides targeting Grik2 mRNA

The Grik2 target inhibitory polynucleotide sequence is designed based on the predicted complementarity with human Grik2 mRNA, such as the Grik2 mRNA sequence of SEQ ID NO: 164, and is synthesized according to methods known in the art. The inhibitory polynucleotide contains a backbone-loop sequence that includes an antisense leader sequence that specifically hybridizes to the Grik2 mRNA region and a sense follower sequence that is essentially complementary to the leader strand. The anti -Grik2 backbone-loop sequence can be transfected into the human neuroblastoma cell line SHSY5Y or the murine N2A neural cell line. Total RNA can be extracted and small RNA sequenced to quantify leader and follower strand concentrations and identify Drosha and Dicer cleavage sites. To measure Grik2 attenuation efficacy, the constructs were co-transfected with human Grik2 cDNA into HEK293 cells, the mRNA was extracted, and real-time quantitative polymerase chain reaction (RT-qPCR) was performed on Grik2 mRNA. In addition, synthetic backbone-loop RNA can be transfected into SHSY5Y cells, followed by mRNA extraction and Grik2 RT-qPCR. Example 2 : Treatment of epilepsy in human subjects by administration of viral vectors encoding inhibitory polyRNAs encoding one or more target Grik2 mRNAs

Subjects diagnosed with epilepsy (e.g., TLE, such as mTLE or ITLE), such as human subjects (e.g., pediatric or adult subjects), may be treated using the compositions described herein to alleviate one of the symptoms of epilepsy, including But not limited to one or more (such as 2 or more, 3 or more, 4 or more) of the following conditions: (a) risk of seizure recurrence; (b) reduction of CNS excitotoxicity and related nerve cell death; (c) Restore the physiological excitatory-inhibitory balance in affected areas of the CNS system; (d) Reduce one or more symptoms of epilepsy (e.g., frequency, duration, or intensity of seizures, weakness, absence, sudden confusion, difficulty understanding, or production Impaired speech, cognition, mobility, dizziness or loss of balance or coordination, paralysis, and mood disorders), and (e) pathological growth of recurrent mossy fibers in the dentate granule cells of the hippocampus. Methods of treatment may optionally include diagnosing or identifying the subject as a candidate for treatment with the compositions of the present disclosure prior to administration. The composition may include an inhibitory polynucleotide that targets Grik2 mRNA (e.g., a polynucleotide encoding an inhibitory RNA sequence of the present disclosure) or a nucleic acid vector (e.g., a virus) containing the same polynucleotide (e.g., an AAV9 vector). Vectors, such as AAV vectors, for example, having a vector selected from the group consisting 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. AAV vector or lentiviral vector of any serotype of HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16). Exemplary inhibitory polynucleotides have no less than 85% identity with any one of the nucleic acid sequences of SEQ ID NOs: 1-19, 34-62, 97-108, 133-147, 226-229 and 238-241 (e.g. , at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more ( For example, 100%))) sequence identity, or it may have one or more of the sequences of SEQ ID NOs: 1-19, 34-62, 97-108, 133-147, 226-229 and 238-241 (also See Figures 1B-1W, Figures 2B-2Q, Figures 3B-3L, Figures 4B-4F, Figures 5B-5E, and Figures 6A-6B for conceptual diagrams). Additionally, viral vectors (eg, AAV vectors) can incorporate expression cassettes containing inhibitory polynucleotides and regulatory sequences that promote heterologous expression of the polynucleotide, such as promoter sequences (see Tables 11 and 12).

The compositions may be administered to the subject by any suitable means, including, for example, intravenous, intraperitoneal, subcutaneous, or transdermal administration, or by direct administration to the central nervous system of the animal (e.g., stereotaxic, intraparenchymal, intrathecal or intracerebroventricular injection). The composition may be administered in a therapeutically effective amount, such as 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ genome copies per subject Dosage in quantity (GC), dose per kilogram (total weight of subject) 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 8 , ^{10 9 ,} 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ or 10 ¹⁴ GC , administered at 10 ⁵ , 10 ^{6 ,} 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 10 , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ GC per gram of patient's brain mass. Subjects' brain weight estimates were obtained from MRI brain volume measurements, which were converted to brain mass and used to calculate precise drug doses. It is also possible to use published databases to estimate brain weight based on age ranges. The agent may be administered bimonthly, monthly, biweekly, or at least weekly or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times per week). repeatedly). The composition may be administered in combination with a second therapeutic modality, such as a second therapeutic agent (e.g., an anti-epileptic drug), surgical intervention (e.g., surgical resection, radiosurgery, gamma knife or laser ablation), vagus nerve stimulation, deep brain stimulation, or transtranscriptional therapy. Magnetic cranial stimulation.

The composition can be administered to a subject in an amount sufficient to reduce one or more (e.g., 2 or more, 3 or more, 4 or more) of the following symptoms: (a) the risk of recurrence of seizures; (b) reducing Excitotoxicity of the CNS and associated neuronal cell death; (c) Restoration of physiological excitatory-inhibitory balance in affected areas of the CNS; (d) Reduction of one or more symptoms of epilepsy (e.g., seizure frequency, duration, or intensity, weakness , absence of consciousness, sudden confusion, difficulty understanding or producing speech, impaired cognition, difficulty moving, dizziness or loss of balance or coordination, paralysis, and mood disorders), and (e) recurrent mossy fibers of hippocampal gyrus dentate granule cells Pathological growth of 10% or more (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). Symptoms of epilepsy listed above can be evaluated using standard methods, such as neurological examination, electroencephalogram, magnetoencephalogram, CT scan, PET scan, fMRI scan, videography, and visual observation. Measurements of epilepsy symptoms before and after administration of the composition can be compared to evaluate the efficacy of the treatment. The above-mentioned finding of alleviation of epilepsy symptoms indicates that the composition has successfully treated the subject's epilepsy.

Figures 1B-1W, Figures 2B-2Q, Figures 3B-3L, Figures 4B-4F The numbers in black circles correspond to different rationales for the anti -Grik2 construct design of the present disclosure. Therefore, the rationale for each number assignment is as follows: ❶ RISC assembly favors miRNA strands with 5' ends that are more likely to fray or be released from the double helix (Schwarz et al., Cell 115:199-208 (2003); Khvorova et al. , Cell 115:209-16 (2003); and Medley et al., Wiley Interdiscip. Rev. RNA 12:e1627 (2021)). The construct disclosed herein facilitates RISC guide selection by destabilizing the base pairing at the 5' end of the guide strand (introducing a UA pair, a UG wobble pair or mispairing, or deleting a GC pair). ❷ RISC assembly facilitates the miRNA strands with 5' ends to be more easily frayed or released from the double helix. The construct disclosed herein is not conducive to RISC follower selection by introducing GC pairs at the 5' end of the follower strand to tighten base pairing at the 5' end of the follower strand. ❸The 5' terminal nucleotide of the small guide RNA is anchored in the phosphate-binding pocket Argonaute (AGO) protein and cannot base pair with its target RNA (Medley et al., Wiley Interdiscip. RNA 12:e1627 (2021); Ma et al., Nature 434:666-70 (2005); Parker et al., Nature 434:663-6 (2005); and Ghildiyal et al., RNA 16:43-56 (2010)). ❹ 5'-U is the preferred nucleotide for AGO2 binding (Seitz et al., Silence 2:4 (2011); Frank et al., Nature 465:818-22 (2010); De et al., Mol. Cell 50:344 -55 (2013); and Czech et al., Nat. Rev. Genet. 12:19-31 (2011)). Introducing U as the first (5') nucleotide of the leader strand and G or C as the first (5') nucleotide of the follower strand promotes AGO2 binding. ❺ Introducing 3' mispairing between the guide and its target mRNA results in more efficient silencing of abundant mRNAs in mammalian cells (De et al., Mol. Cell 50:344-55 (2013); Bofill-De Ros Methods 103:157- 66 (2016); and Amarzguioui et al., Nucleic Acids Res. 31:589-95 (2003)). Although extensive complementarity in the seed region (guide nucleotides 2-8 (g2-g8)) and the middle region of the guide strand is critical for AGO2-mediated cleavage of the mRNA target, base pairing at the 3' end is not required. Indeed, mispairing of positions g18, g19, g20, and g21 with the target mRNA weakened the release of guide RNA from AGO2, an unloading activity regulated by the target mRNA. ❻ Mispairing of the seed region (leader strand nucleotide positions 2-8) between the leader and follower strands promotes unwinding of the follower strand during RISC loading, thereby promoting RISC maturation (Ghildiyal et al., RNA 16:43-56 (2010); Tomari et al., Cell 130:299-308 (2007); Matranga et al., Cell 123:607-20 (2005); and Kawamata et al., Nat. Struct. Mol. Biol. 16:953-60 (2009)). The constructs disclosed herein promote leader-RISC complex maturation by introducing mismatches in the seed region of the leader strand, or by converting mispairs into pairs in the corresponding regions of the follower strand (follower strand nucleotide positions 2-8) nucleotides to reduce or prevent the maturation of the companion-RISC complex. ❼ Introduce GC or UA base pairing to replace the UG swing at the junction of the backbone region and the stem-loop region to promote Dicer protein cleavage (Liu et al., Cell 173: 1191-203 (2018)). ❽ Introducing GC base pairing in the flanking region near the backbone-loop structure (i.e., Drosha cleavage site) can help improve the accuracy of Drosha cleavage. ❾ Introducing paired or mispaired base pairs in the backbone region by mimicking the scaffold structure of E-miR-124. Example 3 : In vitro attenuation of Grik2 mRNA using synthetic inhibitory polynucleotides

To determine the attenuated in vitro efficacy using inhibitory polynucleotides of the present disclosure, backbone-loop RNA was synthesized by Integrated DNA Technologies and reconstituted in nuclease-free duplex buffer. After incubation at 95°C for 2 minutes and subsequent gradual cooling to room temperature, intramolecularly bound oligonucleotides were obtained. The siRNA negative control (siNegative, Cat.# AM4621) and two positive control siRNAs targeting Grik2 (siPositive-1 and siPositive-2, Cat.#4392420 and 4392420) were obtained from Life Technologies. SH-SY5Y cells were transfected with 10 nM adhesive backbone-loop RNA oligonucleotides or siRNA using LIPOFECTAMINE™ RNAiMAX Transfection Reagent according to the manufacturer's protocol. Transfection mix without RNA (RNAiMAX only) served as a further transfection control. Transfections were performed in quadruplicate. Cells were further cultured at 37°C, 5% CO until 72 h post- _transfection before cell lysis and qRT-PCR. Expressed concentrations of Grik2 mRNA were normalized to actin (ACTB). Relative Grik2 performance concentrations were obtained by comparison with the siNegative control set to 100%.

All tested backbone-loop RNA oligonucleotides as well as the positive control showed attenuation of Grik2 mRNA in SH-SY5Y cells compared to the negative control and transfection reagent alone (RNAiMAX) ( Figure 7 ). Statistically significant attenuation rates were achieved at 72 hours post-transfection (remaining mRNA represented between 42% and 69%). Compared to miRNA constructs A, C and D, the tested constructs maintained Grik2 attenuating efficacy. Example 4 : RNA sequencing indicates favorable microRNA processing properties in mouse neurons.

Embedding the backbone-loop sequence (i.e., E-miR-30, E-miRNA-124-3, or E-miR-218-1) in the endogenous microRNA scaffold, and encoding the AAV genome (pro-AAV) Secondary selection occurs in cis-plastids and under the regulatory control of the neuron-specific promoter hSyn1 (Figures 5A-5E). Plasmids were transfected into N2A mouse neuronal cells. Mouse N2A neuroblasts were seeded into 24-well plates at 9.0E4 cells per well, and 24 hours later the cells were transfected using 250ng plastid and 1.2μl Lipofectamine 3000 per well. After 48 hours, small RNA-enriched RNA was isolated using the Qiagen miRNeasy mini kit, and library preparation and small RNA sequencing were performed. A microRNA sequencing library was generated using the NEXTFLEX® small RNA kit from Perkin Elmer/BIOO. Sequencing was performed on the MiSeq platform.

SH-SY5Y cells were transfected by electroporation. Total RNA was extracted with Trizol, and a microRNA sequencing library was generated using the NEBNEXT® kit from New England Biolabs (NEB). Sequencing was performed on Novogene’s HiSeq.

Rationally designed miRNAs are expressed and processed in mouse N2A neurons after plastid transfection. Rationally designed microRNA constructs exhibit improved leader-to-follower (G:P) ratios compared to the parental sequences (Constructs A, C, and D) ( see Table 14 ). Specifically, for four rationally designed constructs (Construct #1-4) with one or more modifications compared to Construct A, the G:P ratio of Construct A increased from 0.8 to between 11 and 27 The ratio. Benefiting from the reduced entourage and potential improvement in miRNA maturation, the guide strand concentration (percent guide strand count in the total miRNA library) was increased from 0.25% for constructs #1-4 to 1.3-2.6%. In constructs #39, #40, and #41, the G:P ratio of construct C was 1 and the primer concentration was 2.8%, increasing to over 100 and 5%-21%, respectively. Constructs #50 and #51 exhibited improved G:P ratios above 2000 relative to construct D's G:P ratio of 1. Finally, the construct #51 bootstrap concentration was increased to 5.1%.

Similar results were observed using the human neuroblastoma cell line SH-SY5Y. The G/P ratio of GI in both the original construct and the redesigned construct was higher than that observed in N2A cells. This may be due to differences in biogenesis/degradation constants in the two cell systems, or it may be due to the different sets used for sequencing library preparation. Table 14. Improving the leader/follower ratio Parental guidance and scaffolding construct N2A cells SH-SY5Y cells * G:P ratio guide concentration G:P increase ( collapse ) Boot increase ( collapse ) G:P G:P increase ( collapse ) GI, E-mir30a Construct A SEQ ID NO: 1 0.8 0.25% 30.3 Construct #1 (SEQ ID NO: 2) 10 2.66% 12.5 10.6 782.5 25.8 Construct #2 (SEQ ID NO: 3) 13 1.98% 16.2 7.9 941.6 31.1 Construct #3 (SEQ ID NO: 4) twenty three 2.10% 28.8 8.4 912.2 30.1 Construct #4 (SEQ ID NO: 5) twenty four 1.33% 30 5.3 616.9 20.4 MW, E-mir218-1 Construct D (SEQ ID NO: 133) 5.8 0.93% 34 Construct #50 (SEQ ID NO: 134) 529 0.42% 91 0.45 217 6.4 Construct #51 (SEQ ID NO: 135) 1863 5.11% 321 5.5 755 22.2 MW, E-mir124-3 Construct C (SEQ ID NO: 97) 1 2.83% 0.25 Construct #39 (SEQ ID NO: 98) 121 19.92% 121 7.0 1572 6288 Construct #40 (SEQ ID NO: 99) 12312 4.97% 12312 1.8 NP** Construct #41 (SEQ ID NO:100) 535 21.3% 535 7.5 8296 33184 G9, E-mir124-3 Construct B (SEQ ID NO:80) 0.01 0.30% 0.0015 Construct #23 (SEQ ID NO:81) 0.72 0.39% 72 1.3 0.016 10.7 Construct #24 (SEQ ID NO:82) 0.12 0.21% 12 0.7 0.0078 5.2 Construct #31 (SEQ ID NO: 89) - - - - 0.0657 43.8

Two redesigned constructs #3 (GI, E-miR-30a) and #51 (MW, E-miR218-1) were selected, and two redesigned constructs #100 ( Figure 6A , SEQ ID NO: 256) and #101 ( Figure 6B , SEQ ID NO: 257) were concatenated in two different orders. Compared to single constructs #102 ( Figure 5B ) and #103 ( Figure 5C ), a strong clustering effect was observed in both concatemer constructs in N2A cells, with enhanced MW and suppressed GI. However, the total yield of GI and MW produced by either concatemer was higher than the total yield of GI and MW produced alone in a single construct carrying the filler sequence (Table 15). Table 15. Sequencing analysis of redesigned concatemer constructs in N2A cells Construct ( in plastid ) GI G:P GI%* MW G:P MW%* #102 (#3 and filler sequence, SEQ ID: 252)) 37.4 0.7% #103 (#51 and filler sequence, SEQ ID: 253) 375.0 0.8% #100 (#3 + #51 and filler sequence, SEQ ID: 256) 26.8 0.1% 581.0 2.4% #101 (#51 + #3 and filler sequence, SEQ ID: 257) 61.8 0.4% 690.0 6.9% * Bootstrap count / total miRNA library

From the original four constructs (Construct A ( Table 16 ), Construct B ( Table 17 ), Construct C ( Table 18 ), and Construct D ( Table 19 )), 42 additional redesigns were generated. To reduce the number of transfected and sequenced samples, perform co-transfection of 2-3 accessions in N2A cells (e.g., co-transfection of constructs #7, #42, and #27 in Tables 16 , 17 , and 18 stain), and then perform small RNA sequencing. Most of these redesigns exhibit improved leader/follower ratios relative to the original construct. In particular, Construct B, which contains the G9 leader, has a leader/follower ratio below 0.01, which is significantly improved by changes resulting in construct #36 ( Table 17 ), which exhibits a leader/follower ratio of 90.5. In the scaffold of hsa-mir-30a, four redesigns encoding GI showed improved leader/follower ratios of over 100 ( Table 16 ). The percentage of primers in the total miRNA pool may be underestimated for each redesigned construct due to co-transfection with plasmids in the sample. Table 16. Redesign of GI from hsa-mir-30a scaffold of construct A construct boot % * G/P ratio Number of co-transfected plasmids #3 0.9% 26.6 1 #3 1.8% 22.4 1 #5 0.89% 85.2 3 #6 0.89% 16.8 3 #7 1.1% 25.8 3 #8 0.61% 55 3 #9 0.72% 78 3 #10 3.2% 50 3 #11 0.26% no entourage 3 #12 1.2% 105 3 #13 0.32% no entourage 3 #14 1.0% 22.6 3 #15 0.89% 22.6 2 #16 0.38% 49.1 3 #17 0.9% 34.8 1 #18 0.5% 104 1 * Boot Count / Total miRNA Library Constructs #19 , #20 , #21 and #22 have similar redesign characteristics to #11 and #13 in the table and were not performed in plasmid transfection and small RNA sequencing analysis Experimental testing. Table 17. Redesign of G9 from the hsa-mir-124-3 scaffold of construct B construct boot % * G/P ratio Number of co-transfected plasmids B 0.24% 0.0038 1 #25 1.3% 1.6 3 #26 1.0% 0.64 3 #27 1.0% 1.1 3 #28 0.44% 0.2 3 #29 0.49% 0.3 3 #30 0.48% 0.16 3 #35 0.22% 3.7 3 #36 0.65% 90.5 2 * Boot count / total miRNA library Table 18. Redesign of MW from hsa-mir-124-3 scaffold of construct C construct boot % * G/P ratio Number of co-transfected plasmids C 1.7% 0.63 1 #39 17.9% 99 1 #40 11.7% no entourage 1 #42 14.3% 838 3 #43 10.3% 75 3 #44 11.5% 782 3 #45 13.1% 666 3 #46 17.6% 271 3 #47 12.4% 1947 3 #48 9.7% 60 3 #49 7.5% 393 3 * Boot count / total miRNA library Table 19. Redesign of MW from hsa-mir-218-1 scaffold of construct D construct boot % * G/P ratio Number of co-transfected plasmids #51 3.1% 1192 1 #51 8.5% 899 1 #52 0.06% 44 3 #53 0.6% 139 3 #54 4.4% 884 3 * Bootstrap count / total miRNA library Example 5 : GLUK2 protein was significantly reduced in mouse cortical neuronal cells after treatment with an AAV9 vector encoding a redesigned concatemer .

For protein collection, dissociated cortical neurons from P0-P1 C57Bl6/J mice were prepared and neurons were seeded in six-well dishes at a concentration of 5.5e+5 cells per well. Two or three days after inoculation (days in vivo, DIV2-3), remove half of the culture medium and add virus at an MOI of 7.5E+4. At DIV 13, mouse neuronal cultures were lysed, and lysates were used for SDS PAGE and immunoblotting. For immunostaining, the following antibodies were applied: rabbit anti-GluK2/3 (clone NL9 04-921; Merck-Millipore) and mouse anti-β-actin (A5316; Sigma) as primary antibodies, and appropriate antibodies produced in goat 800nm fluorescent-conjugated secondary antibodies (IRDye 800 goat anti-mouse Li-COR 926-32210 or IRDye 800 goat anti-rabbit Li-COR 926-32211) were used as secondary antibodies. Target proteins are detected by reading at 800nm on Li-COR. Analyzes were performed using Empiria Studio software. For quantitation, fluorescent signal intensity in each lane was normalized by β-actin expression and then by control conditions.

For RNA collection and miRNA quantification by backbone-loop RT-qPCR, mouse cortical cultures were rinsed in ice-cold PBS at DIV 13 and scraped into 700 µl Qiazol (QIAGEN). Total RNA was extracted using the miRNeasy mini kit (QIAGEN 217004). The total RNA content and quality of the samples were assessed by a NANODROP™ One spectrophotometer (Thermofischer). Reverse-transcribe 20ng of total RNA or synthetic oligoRNA (10 ³ - 10 ⁹ copy number) into cDNA using the TaqMan microRNA Reverse Transcription Kit and GI or MW sequence-specific reverse transcription primers, then use TAQMAN ^TM Fast Universal qPCR Master Mix and specific Perform qPCR using GI or MW qPCR primers/probes. The number of copies of GI or MW in a sample calculated from a standard curve established using synthetic GI or MW oligoRNA. result

Concatemer vectors #100 and #101 (SEQ ID NO: 256 and 257, respectively) yielded GI and MW both, thus producing higher concentrations of therapeutic miRNAs in MCN cells ( Figure 8A ). A cluster effect may exist, as MW performance appears to be enhanced when linked to GI in the same transcript. The negative control AAV9 RNA null vector (Ctrl) carries a stuffer sequence in the vector genome but does not contain any miRNA construct. Consistently, a significant decrease in GLUK2 was observed in MCN samples treated with concatemer vectors #100 and #101, but not in single construct vectors ( Figure 8B ). Example 6 : Grik2 transcripts are significantly reduced in human GlutaNeuron cells following treatment with an AAV9 vector encoding a redesigned concatemer .

Thaw iPSC-induced ICELL® GlutaNeurons (Fujifilm Cat.R1034) and seed cells into PLO/Matrigel-coated 6-well dishes at a density of 2E+6 cells per well. After 24 hours, AAV9 vector #100 or RNA null vector #106 (SEQ ID NO: 262) was added to cells at an MOI of 3E+5 in 2 mL of fresh medium (n = 4 per condition). Eleven days after trait introduction, cells were harvested by adding Qiazol lysis buffer and adding RNA oligonucleotides (3uL 0.1nM nine-oligonucleotide mixture per 50,000 cells). Total RNA was extracted and enriched for miRNA following the protocol for isolating large RNA and small RNA/miRNA-enriched fragments using the Qiagen miRNeasy kit. Generate microRNA sequencing libraries using Perkin Elmer/BIOO's NEXTFLEX® Small RNA Kit. Sequencing was performed on the HiSeq platform (Genewiz). An RNA-seq library was also prepared and sequenced.

Small RNA sequencing analysis showed that synthetic miRNA GI and MW were processed at physiologically active concentrations with high leader/follower ratios in Construct #100-treated iPSC-derived GlutaNeuron cells ( Table 20 ). At a trait import efficiency of 50%, Grik2 transcripts were reduced by 18.5% in #100-treated iPSC-derived GlutaNeuron cells (n=4) ( Figure 9 ). Table 20. Small RNA sequencing of iPSC- derived GlutaNeuron cells introduced using #100 trait # 100Sample 1 2 3 4 average GI boot count 2613 42656 15518 117694 MW boot count 9538 156031 49625 358452 GI Guide / Cell 423.9 477.7 536.9 567.1 501.4 MW boot / cell 1547.2 1747.5 1717.1 1727.1 1684.7 total boot / cell 1971.1 2225.2 2254.0 2294.2 2186.1 Leader / Follower Ratio - GI 522.6 370.9 316.7 476.5 421.7 Lead / Follow Ratio - MW 733.7 1608.6 1378.5 1525.3 1311.5 Example 7 : Inhibition of seizure activity in human hippocampal organotypic slices introduced using concatemer vector traits.

Human organ slices were individually transferred to a recording chamber maintained at 30 to 32°C and continuously perfused (each time) with oxygenated (95% O2 and 5% CO2) ACSF in the presence of 5 µM gabazine (Sigma-Aldrich). min 2 to 3 mL); contains ACSF (in mM): NaCl (126.0), KCl (3.5), NaH ₂ PO ₄ (1.2), NaHCO ₃ (26), MgCl ₂ (1.3), CaCl ₂ (2.0 ) and glucose (10), pH approximately 7.4 (Sigma-Aldrich). Local field potential (LFP) recordings were made using monopolar nichrome wires placed in the granule cell layer of the dentate gyrus. A DAM-80 amplifier was used for recording (low filter, 0.1 Hz; high-pass filter, 3 KHz; World Precision Instruments, Sarasota, FL); data were digitized (20 kHz) to a computer using a Digidata 1440A (Molecular Devices), and Obtained using Clampex 10.1 software (PClamp, Molecular Devices). Signals were analyzed offline using Clampfit 9.2 (PClamp) and MiniAnalysis 6.0.1 (Synaptosoft, Decatur, GA). result

Concatemer Vector #100 (vector diagram depicted in Figure 14 ) inhibits TLE hippocampal spontaneity in vitro under hyperexcitable conditions in the presence of 4-AP/gabazine ( Figure 10A ) and under physiological conditions ( Figure 10B ). Example 8 : Physiological and behavioral studies in a pilocarpine epilepsy mouse model indicated an improvement in epileptic symptoms. AAV cast

Male Swiss mice previously rendered epileptic by systemic injection of pilocarpine for at least 2 months were placed in a stereotaxic frame. Four holes were drilled and AAV9 was injected bilaterally into the dorsal and ventral dentate gyrus of the hippocampus. Slowly inject the defined volume of solution containing AAV (1.0 µL 2.5e8 GC per injection site, two injection sites per hemisphere, in this way 5e8 GC per hemisphere and 1e9 per brain) at a rate of 0.2 µl per minute. Hyperactivity

Movement of epileptic mice (>2 months post-SE) was assessed 1 week before and 2 weeks after AAV injection ( Fig. 11A ). Movement of non-epileptic mice (wild-type Swiss male mice, 18 to 21 weeks old) was also assessed. Mice were transferred to the behavioral analysis room 1 day before the experiment to acclimate; mice were housed at room temperature (20 to 22°C) with a 9:00 to 18:00 light/dark cycle and had ad libitum access to food and water. Thereafter, all materials that had been in contact with the test animals were cleaned with acetic acid to prevent olfactory cues. First, spontaneous exploratory behavior was tested using an open field experiment (Müller et al., 2009). Briefly, mice were placed into the center of a 50 × 50 × 50 cm blue polyvinyl chloride box for 10 min, and trajectories were recorded using a video camera connected to the tracking software EthoVision Color (Noldus, The Netherlands); mice were analyzed Speed and total distance during the 10-minute exploration period. EEG electrode implantation and recording

A depth lead electrode was implanted into the treated mice 3 weeks after AAV injection. The surgery is performed under isoflurane anesthesia. Electrodes were placed stereotaxically into the dentate gyrus (DG) (Paxinos and Watson coordinates from bregma: AP -2.55 mm, ML +1.65 mm, DV -2.25 mm). Place an additional screw above the cerebellum to serve as a ground electrode. Dental cement is used to secure the electrodes and screws to the skull. During the recovery period, animals will be given 5 mg s.c. carprofen (RIMADYL®) per kilogram after 24 and 48 hours.

EEG was monitored 24 h per day for 5 days using a telemetry system (Data Sciences International, St. Paul, MN) (magnified (1000x), filtered at 0.16 to 97 Hz, acquired at 500 Hz).

The intrahippocampal EEG trace represents the potential difference between the electrode inserted in the DG and the electrode located above the cerebellum. Small RNA sequencing analysis:

Four weeks later, the animals were sacrificed, and the hippocampi were excised and snap frozen. After lysis, total RNA was extracted from the same mouse hippocampus tissue using the ALLPREP® DNA/RNA Mini Kit (Qiagen, REF# 80204). Use the NEXTFLEX® small RNA sequencing v3 kit (NOVA-5132-22) with Illumina UDI in GenomeScan to obtain 400ng of total RNA and newly add RNA oligonucleotide mixture (4500 molecules per 10pg of total RNA) for data Library preparation. Possible adapter dimers were removed using Ampure magnetic bead size selection. Clustering and DNA sequencing were performed using NovaSeq6000 according to the manufacturer's protocol. result

Construct #100 and Construct #101 effectively reduced the hyperactive phenotype in the pilocarpine model in vivo at the tested dose of 1e9 GC per brain ( Figure 11A ). All other constructs, including the RNA null control construct (#106) and the two single constructs, did not reduce hyperactivity after treatment. EEG evaluation further showed that Construct #100 and Construct #101 were effective in reducing the average number of seizures per day compared to the control group ( Figure 11B ). Based on these results, it appears that the concatemer design with one promoter driving the expression of both miRNAs found in construct #100 and construct #101 has better performance than that found in construct #102 and construct #103. A single design in which one promoter drives the expression of one miRNA is more efficient.

Three operators independently scored five animal behaviors (nesting, rocking, grooming, touching, and locomotion). Mice treated with the Grik2 targeting vector had improved behavior compared to mice with pilocarpine-induced epilepsy treated with the RNA null control vector #106, with construct #100 exhibiting the most significant effect ( Figure 11C ).

Total GI and MW copy numbers expressed and processed from construct #100 AAV9 vectors injected bilaterally into the mouse hippocampus were measured by small RNA sequencing analysis. Per 10 pg of total RNA (average amount per cell), 392.2 and 573.1 copy numbers of GI and 1331.3 and 2138.8 copy numbers of MW were detected in the two hippocampal gyri, respectively ( Table 21 ). The molecules of miRNA are within the range of physiological activity. Leader/follower ratios above 100 for both GI and MW indicate less than 5 copy numbers per 10pg of follower strands and are below physiologically active concentrations. Table 21. Small RNA sequencing of two hippocampal gyri treated with Concatemer #100 right left GI guide copy number/cell 392.2 573.1 MW guide copy number/cell 1331.3 2138.8 Total miRNA copy number/cell 1723.5 2711.9 Leader/Follower Ratio - GI 435.4 292.7 Lead/Follow Ratio - MW 369.5 241.0

To further evaluate the effectiveness of these microRNA constructs in reducing epileptic symptoms in the brains of pilocarpine epileptic mice, the injection dose was modified to 2.5 x 10 ⁹ GC per injection site, with 2 injection sites per hemisphere, in After performing a dose response study, the results were 5 x 10 ⁹ GC per hemisphere and 1 x 10 ¹⁰ GC per brain ( Figure 12A and Figure 12B ). Likewise, Construct #100 proved effective in reducing hyperactivity and reducing seizure activity in vivo . In a separate dose-response study, a hyperactive phenotype was observed in pilocarpine mice treated with vehicle #100 at the highest doses of 1E+9 and 1E+10 per brain compared to RNA-null control vehicle. ( Fig. 13A ) and dose-dependent reduction in epileptic seizures ( Fig. 13B ). Other embodiments

Various modifications and variations described in this disclosure will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes for carrying out the disclosure that are apparent to those skilled in the art are intended to be within the scope of the disclosure.

Other embodiments are within the scope of the patent claims. References

Throughout this application, various references describe the prior art to which this disclosure belongs. The disclosures of these references are hereby incorporated by reference into this disclosure: Bahn S., Volk B. Wisden W. (1994). Kainate receptor gene expression in the developing rat brain. (Kainate receptor gene expression in the developing rat brain.) J. Neurosci. 14 5525-5547. 10.1523/JNEUROSCI.14-09-05525.1994. Boudreau Ryan L., Rodríguez-Lebrón Edgardo, Davidson Beverly L., Brain RNAi Medicine: Progress and Challenges, Human Molecular Genetics, Volume 20, Issue R1, 15 April 2011, Pages R21 to R27 Bouvier G., Larsen R.S., Rodríguez-Moreno A., Paulsen O., Sjostrom P.J. (2018). Resolving the presynaptic NMDA receptor debate. (Towards resolving the presynaptic NMDA receptor debate.) Curr.Opin.Neurobiol.51 1-7. 10.1016/j.conb.2017.12.020 Crépel V, Mulle C (2015) Physiological pathology of kainate receptors in epilepsy. (Physiopathology of kainate receptors in epilepsy.) Curr Opin Pharmacol 20:83-88; doi: 10.1016/j.coph.2014.11.012. Epub 2014 Dec 13. Englot, DJ. et al (2013) Seizure outcomes after resection surgery for extratemporal epilepsy in pediatric patients: a systematic review. (Seizure outcomes after resective surgery for extra-temporal lobe epilepsy in pediatric patients: A systematic review.) J. Neurosurgery. 12(2):97-201 Fritsch B., Reis J., Gasior M., Kaminski R. M., Michael A., Rogawski M. A. (2014). Role of the GluK1 kainate receptor in epileptic seizures, epileptic discharges, and epileptogenesis. (Role of GluK1 kainate receptors in seizures, epileptic discharges, and epileptogenesis.) J. Neurosci. 34 5765-5775.10.1523/JNEUROSCI.5307-13.2014 Gabriel S, Njunting M, Pomper JK, Merschhemke M, Sanabria ERG, Eilers A, Kivi A, Zeller M, Meencke H-J, Cavalheiro E a, einemann U, Lehmann T-N (2004) Patients with and without hippocampal sclerosis Stimulation and potassium-induced epileptiform activity in the human dentate gyrus. (Stimulus and potassium-induced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis.) J Neurosci 24:10416-10430. Gruber A., Lorenz R., Bernhart S.H., Neuböck R., Hofacker I.L (2008). Vienna RNA Network Kit. Nucleic acid research. 36:W70-4 Hardy, J. (2010). Genetic analysis of pathways in Parkinson's disease. (Genetic analysis of pathways to Parkinson disease.) Neuron, 68 (2), 201–206. doi:10.1016/j.neuron.2010.10.014 Melyan Z., Lancaster B., Wheal H. V. (2004). Metabolic regulation of intrinsic excitability by synaptic activation of kainate receptors. (Metabotropic regulation of intrinsic excitability by synaptic activation of kainate receptors.) J. Neurosci. 24 4530-4534.10.1523/JNEUROSCI.5356-03.2004 Melyan Z., Wheal H. V., Lancaster B. (2002). Metabotropically mediated kainate receptors regulate isAHP and pyramidal cell excitability. (Metabotropic-mediated kainate receptor regulation of isAHP and excitability in pyramidal cells.) Neuron 34 107-114. 10.1016/S0896-6273(02)00624-4 Mulle C., Sailer A., Pérez-Otaño I., Dickinson-Anson H., Castillo P. E., Bureau I. et al. (1998). Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice. (Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice.) Nature 392 601-605. Peret A, Christie L a., Ouedraogo DW, Gorlewicz A, Epsztein J, Mulle C, Crépel V (2014) Contribution of abnormal GluK2-containing kainate receptors to chronic seizures in temporal lobe epilepsy. (Contribution of Aberrant GluK2-Containing Kainate Receptors to Chronic Seizures in Temporal Lobe Epilepsy.) Cell Rep 8:347-354. Reiner A, Arant RJ and Isacoff EY (2012) Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex. (Assembly Stoichiometry of the GluK2/GluK5 Kainate Receptor Complex.) Cell Rep 1:234-240. Represa A, Le Gall La Salle G, Ben-Ari Y (1989a) Hippocampal plasticity in a rat challenge model of epilepsy. (Hippocampal plasticity in the kindling model of epilepsy in rats.) Neurosci Lett 99:345-350. Represa A, Robain O, Tremblay E, Ben-Ari Y (1989b) Hippocampal plasticity in childhood epilepsy. (Hippocampal plasticity in childhood epilepsy.)Neurosci Lett 99:351-355. Rodríguez-Moreno A., Herreras O., Lerma J. (1997). Presynaptic downregulation of GABA inhibition by kainate receptors in the rat hippocampus. (Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus.) Neuron 19 893-901. 10.1016/S0896-6273(00)80970-8. Rodríguez-Moreno A., Sihra T. S. (2007a). Kainate receptors with metabolic modes of operation. (Kainate receptors with a metabotropic modus operandi.)Trends Neurosci. 30 630-637. Rodríguez-Moreno A., Sihra T. S. (2007b). Metabotropic effects of kainate receptors in the central nervous system. (Metabotropic actions of kainate receptors in the CNS.) J. Neurochem. 103 2121-2135. Sapru Mohan K., Yates Jonathan W., Hogan Shea, Jiang Lixin, Halter Jeremy, Bohn Martha C. (2006). Silencing of human α-synuclein in vitro and in rat brain using lentivirally mediated RNAi. (Silencing of human α-synuclein in vitro and in rat brain using lentiviral-mediated RNAi.)Neurology.198:382-390 Smolders I., Bortolotto Z.A., Clarke V.R., Warre R., Khan G.M., O'Neill M.J., et al. (2002). Antagonists containing GLU (K5) kainate receptors prevent pilocarpine-induced limbic seizures. (Antagonists of GLU(K5)-containing kainate receptors prevent pilocarpine-induced limbic seizures.) Nat. Neurosci. 5 796-804.10.1038/nn88 Sutula T, Cascino G, Cavazos J, Parada I, Ramirez L (1989) Mossy fiber synaptic reorganization in the epileptic human temporal lobe. (Mossy fiber synaptic reorganization in the epileptic human temporal lobe.) Ann Neurol 26:321-330. Tauck DL, Nadler J V (1985) Evidence for rapid growth of functional mossy fibers in hippocampal structures in kainic acid-treated rats. (Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats.) J Neurosci 5:1016-1022 Valbuena S., Lerma J. (2016). Hidden biology of non-canonical signaling, ligand-gated ion channels. (Non-canonical signaling, the hidden life of ligand-gated ion channels.) Neuron 92 316-329. 10.1016/j.neuron.2016.10.016 Wang L., Bai J. and Hu Y. (2007) Identification of RA-responsive elements and transcriptional reticence in the human alphaCaMKII promoter. (Identification of the RA Response Element and Transcriptional Silencer in Human alphaCaMKII Promoter.) Mol.Biol.Rep.35(1):37-44 Zinn, E., Pacouret, S., Khaychuk, V., Turunen, H. T., Carvalho, L. S., Andres-Mateos, E., … Vandenberghe, L. H. (2015). An effective gene therapy vector produced by reconstructing the evolutionary lineage of viruses through computer simulation. 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This patent or application file contains at least one drawing executed in color. Upon application and payment of the necessary fees, the Intellectual Property Office will provide a copy of this patent or patent application publication with a color drawing. Figures 1A to 1W are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence GI (SEQ ID NO: 16) embedded in an endogenous (E)-miR-30 microRNA scaffold or its variations. The stem-loop structure contains: from 5' to 3, the guide strand, which contains the GI antisense sequence or a rationally designed variant thereof (SEQ ID NO: 17 to SEQ ID NO: 30, SEQ ID NO: 230 to SEQ ID NO : 233 and SEQ ID NO: 242 to SEQ ID NO: 245); E-miR-30 loop sequence; and follower sequence (SEQ ID NO: 31) or rationally designed variants thereof (SEQ ID NO: 32 to SEQ ID NO: 45, SEQ ID NO: 234 to SEQ ID NO: 237 and SEQ ID NO: 246 to SEQ ID NO: 249). The starting construct (Construct A) is shown in Figure 1A. Changes relative to the starting construct are shown in Figures 1B-1W respectively. The small black dots correspond to the UG swing pairs. The large black dots with numbers correspond to the design basis 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 that was delivered to induced potentiated stem cells (iPSC) for derivation of glutamatergic neurons (GlutaNeuron). Figures 2A through 2Q are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence G9 (SEQ ID NO: 63) embedded in the endogenous E-miR-124-3 microRNA scaffold or its variations. The stem-loop structure contains: a guide strand, which contains the G9 antisense sequence or a rationally designed variant thereof (SEQ ID NO: 64 to SEQ ID NO: 79); the E-miR-124-3 loop sequence; and the follower sequence ( SEQ ID NO: 80) or rationally designed variants thereof (SEQ ID NO: 81 to SEQ ID NO: 96]. The starting construct (Construct B) is shown in Figure 2A. Relative to the starting construct The changes are shown in Figures 2B-2Q, respectively. The constructs shown in Figures 2A to 2I are characterized by a stem-loop structure containing: from 5' to 3', a follower strand, a loop sequence and a leader strand; and that shown in Figure 2J The construct in 2Q is characterized by a stem-loop structure, which contains: from 5' to 3', the leader strand, the loop sequence and the follower strand. The small black dots correspond to the UG swing pairs. The large black dots with numbers correspond to Design baseline described in Example 1. *Drosha and Dicer cleavage sites were based on the most abundant species observed in small RNA sequencing data obtained from starting construct A, which was delivered to GlutaNeuron Center. Figures 3A to 3L are images of stem-loop structures containing the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 109) embedded in the endogenous E-miR-124-3 microRNA scaffold. ) or a variant thereof. The stem-loop structure contains: from 5' to 3', the following sequence (SEQ ID NO: 121) or its rationally designed variant (SEQ ID NO: 122 to SEQ ID NO: 132); E -miR-124-3 loop sequence; and a guide strand containing the MW antisense sequence or a rationally designed variant thereof (SEQ ID NO: 110-120). The starting construct (Construct C) is shown in Figure 3A Center. Changes relative to the starting construct are shown in Figures 3B-3L, respectively. Small black dots correspond to UG swing pairs. Large black dots with numbers correspond to the design baseline described in Example 1. *Drosha and Dicer cutting sites Points are based on the most abundant species observed in small RNA sequencing data obtained from starting construct A, which was delivered into GlutaNeuron. Figures 4A to 4F are images of stem-loop structures. The stem-loop structures contain the Grik2 mRNA-targeting antisense sequence MW (SEQ ID NO: 139) or a variant thereof embedded in an endogenous (E)-miR-218-1 microRNA scaffold. The stem-loop structures contain: from 5' to 3, guide strand, which contains the MW antisense sequence or a rationally designed variant thereof (SEQ ID NO: 140 to SEQ ID NO: 144); the E-miR-218-1 loop sequence; and the follower sequence ( SEQ ID NO: 145) or rationally designed variants thereof (SEQ ID NO: 146-150). The starting construct (Construct D) is shown in Figure 4A. Changes relative to the starting construct are shown in Figures 4B-4F respectively. The small black dots correspond to the UG swing pairs. The large black dots with numbers correspond to the design basis 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, which was delivered into GlutaNeuron. Figures 5A to 5E are images of AAV expression constructs containing single microRNA constructs of the present disclosure. The general construct architecture is characterized by: from 5' to 3', the AAV 5'ITR; the hSyn1 promoter sequence; the stem-loop sequence, which contains: from 5' to 3', the 5' microRNA flanking sequence, containing the guide strand or 5' stem-loop arm of the follower strand sequence, microRNA (E-miR) loop sequence, 3' stem-loop arm containing the follower strand or leader strand sequence, and 3' flanking sequence; polyadenylation sequence (RGB polyA); and the AAV 3′ ITR (Fig. 5A ). Figure 5B shows an AAV vector, construct #102, reconstituted with the stem-loop sequence of construct #3 (SEQ ID NO: 4). Figure 5C shows an AAV vector, construct #103, reconstituted with the stem-loop sequence of construct #51 (SEQ ID NO: 135). Figure 5D shows an AAV vector containing a reconstruction of the stem-loop sequence of construct #39 (SEQ ID NO: 98). Figure 5E shows an AAV vector reconstructed from the stem-loop sequence of construct #40 (SEQ ID NO: 99). Figures 6A and 6B are images of AAV expression constructs containing concatemer constructs of the present disclosure. Figure 6A shows a dual microRNA AAV vector, construct #100, which contains the stem-loop sequence of construct #3 (SEQ ID NO: 4) and construct #51 (SEQ ID NO: 135), where construct #3 Positioned at 5' relative to construct #51. Figure 6B shows a concatemer AAV vector containing the stem-loop sequence of construct #3 (SEQ ID NO: 4) and construct #51 (SEQ ID NO: 135), where construct #3 is positioned relative to construct Body #51 of 3'. Figure 7 is a graph depicting the relative expression levels of human Grik2 mRNA in SH-SY5Y cells transfected as shown in Example 3, as quantified by RT-qPCR. n = 4 for all cohorts. One-way analysis of variance, Dunnett's multiple comparison test (relative to siNegative). **p <0.001; error bars: standard deviation. Search table: 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. Figures 8A and 8B are graphs respectively showing the expression of miRNA GI and MW and GLUK2 protein levels in mouse cortical neurons (MCN) after transduction with AAV vectors. Figure 8A shows GI and MW quantification 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 AAV vectors: from left to right, RNA null vector (Ctrl); double-miRNA concatemer; construct #100 ( Seq ID: 256), which contains the stem-loop sequence of construct #3 (SEQ ID NO: 4), which is located 5' relative to construct #51 (SEQ ID NO: 135); dual-miRNA poly Conjoint; Construct #101 (SEQ ID: 257), which contains the stem-loop sequence of Construct #51, which is positioned 5' relative to Construct #3; Single construct, which contains 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 immunoblotting. Control wells were treated with AAV9.hSyn.GFP, RNA null control vector or untreated. The graph shows the fold change in GLUK2/GLUK3 performance normalized to β-actin compared to the AAV9.hSyn.GFP control under each condition. **P<0.01. Figure 9 is a graph showing Grik2 mRNA expression quantified by RNA sequencing in iPSC-derived GlutaNeuron cells after transduction with an RNA null vector (Ctrl) or an AAV encoding a dual-miRNA concatemer. That is, construct #100 (SEQ ID NO: 256), which contains the stem-loop sequence of construct #3 (SEQ ID NO: 4) positioned relative to construct #51 (SEQ ID NO: 135 ) is rebuilt. TPM, transcripts per million. **FDR (P adj) < 0.01. Figures 10A and 10B are graphs showing epileptiform activity in adjacent human brain slices from two patients with temporal lobe epilepsy (TLE). Brain slices from one patient were recorded under hyperexcitable conditions and from another patient 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 figure shows the original trace of seizure events recorded after transduction with a control vector (AAV9.hSyn.GFP). The right side of the figure shows the original trajectory depicting epileptiform discharges after transduction with Construct #100 (AAV9.hSyn. Construct #3/Construct #51; SEQ ID NO: 256). Construct #100 significantly inhibited spontaneous seizures in isolated TLE hippocampal gyrus under hyperexcitable conditions compared to control. Figure 10B shows neuronal excitability in organotypic hippocampal slices from another TLE patient; these slices were recorded under physiological conditions to document spontaneous epilepsy after transduction with RNA null control and construct #100 Paroxysmal activity. Construct #100 significantly inhibited spontaneous seizures in isolated TLE hippocampus under physiological buffer conditions compared to control. Figures 11A - 11C are graphs depicting behavioral assessment of epilepsy-related phenotypes in a pilocarpine mouse model. Chronic epilepsy mice with RNA null control vector (Ctrl) or construct #100 (SEQ ID NO: 256), construct #101 (SEQ ID NO: 257), containing only the GI sequence (SEQ ID NO: 252) Single construct and single construct treatments (n=5) 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 concatemer vectors, construct #100 and construct #101, effectively ameliorated epilepsy-related phenotypes in the pilocarpine model in vivo. Figure 11A shows the total distance covered by chronic epileptic mice during 10 min of exploration in the open field box. Relative to non-epileptic mice, epileptic mice were hyperactive and traveled approximately twice as far. According to this, mice treated with the concatemer vector behaved more like non-epileptic mice and traveled shorter distances after treatment. Figure 11B shows the average number of daily seizures in chronic epileptic mice treated with RNA null control, first concatemer construct #100, or second concatemer construct #101. Figure 11C shows behavioral scores based on five animal behaviors (nesting, rocking, grooming, handling, and locomotion). The Y-axis represents the sum of the ratings for the five behaviors. Controls represent epileptic mice treated with control vehicle. Mice treated with Construct #100 exhibited behaviors similar to normal, non-epileptic mice. Figures 12A and 12B are graphs of distance traveled or seizure activity in pilocarpine mice treated with: RNA null control vector (Ctrl) or reconstruction in Headset #100 (SEQ ID NO: 256). At doses tested at 1E+10 GC/brain, Construct #100 effectively reduced the hyperlocomotion phenotype and seizure activity in the pilocarpine mouse model in vivo. Figure 12A shows the total distance covered by chronic epileptic mice during 10 min of exploration in the open field box. Chronic epileptic mice were treated with control vector or construct #100 administered at 1E+10 GC/brain. ****p<0.0001, Mann-Whitney test. Figure 12B shows the average number of daily seizures in chronic epileptic mice one month after treatment with control vehicle or construct #100. **p<0.01, Mann-Whitney test. Figures 13A and 13B are graphs depicting dose-dependent reductions in hyperlocomotion phenotype and seizures in pilocarpine mice treated with Construct #100. Figure 13A shows the total distance covered during 10 minutes of exploration in the open field box. Chronic epileptic mice were treated with RNA null control vector (Ctrl) or construct #100 (1E+8/ 1E+9/ 1E+10 GC/brain). **p<0.01, Mann-Whitney test. Historical locomotor activity of wild-type mice (WT) was assessed in separate experiments but is shown here for comparison. Figure 13B shows the average number of seizures per day in chronic epileptic mice after treatment with control vehicle or Construct #100. Figure 14 is an image of a vector map including the inhibitory polynucleotide sequence of construct #100. Although construct #100 includes the lac promoter sequence, the ampicillin resistance (AmpR) promoter sequence, and the konmycin resistance (KanR) sequence, other promoters and antibiotic resistance sequences may be included (e.g., chloramphenicol resistance sequences) as an alternative.

TW202346585A_112100490_SEQL.xml

Claims (242)

An isolated polynucleotide that specifically binds Grik2 mRNA comprising a stem-loop region including a 5' arm (5p), a loop region and a 3' arm (3p), wherein the stem-loop region The stem-loop region includes a guide strand sequence and a passenger strand sequence, and the guide strand sequence and passenger strand sequence include: (a) Uracil (U)-adenine (A) base pair or U-guanine (G) base pair at the 5' end of the leading strand; (b) Cytosine (C)-G pair at the 5′ end of the follower strand; (c) A U at the 5' end of the leader sequence; (d) A mismatch in the seed region between the leader sequence and the follower sequence; and/or (e) Replace the U-G wobble with C-G base pairs or U-A base pairs at the junction of the stem region and the loop region of the polynucleotide. The polynucleotide of claim 1, wherein (a) and (c) improve a guide strand sequence loaded into an RNA-induced silencing complex (RISC) protein. The polynucleotide of claim 1 or 2, wherein (b) impairs a follower strand sequence loaded into a RISC protein. The polynucleotide of any one of claims 1 to 3, wherein (d) facilitates decoupling of the follower strand sequence from the leader strand sequence during RISC loading. The polynucleotide of any one of claims 1 to 4, wherein (e) the loop region is enhanced by cleavage of the stem region by Dicer. The polynucleotide of any one of claims 1 to 5, wherein the seed region of the leader sequence includes nucleotides 2 to 7 of the leader sequence. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 2. Such as the polynucleotide of claim 7, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 17. Such as the polynucleotide of claim 7 or 8, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 32. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 3. Such as the polynucleotide of claim 10, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 18. Such as the polynucleotide of claim 10 or 11, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 33. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 4. Such as the polynucleotide of claim 13, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 19. Such as the polynucleotide of claim 13 or 14, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 34. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 5. Such as the polynucleotide of claim 16, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 20. Such as the polynucleotide of claim 16 or 17, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 35. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 6. Such as the polynucleotide of claim 19, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 21. Such as the polynucleotide of claim 19 or 20, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 36. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 7. Such as the polynucleotide of claim 22, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 22. Such as the polynucleotide of claim 22 or 23, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 37. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 8. Such as the polynucleotide of claim 25, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 23. Such as the polynucleotide of claim 25 or 26, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 38. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 9. Such as the polynucleotide of claim 28, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 24. Such as the polynucleotide of claim 28 or 29, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 39. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 10. Such as the polynucleotide of claim 31, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 25. Such as the polynucleotide of claim 31 or 32, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 40. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 11. Such as the polynucleotide of claim 34, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 26. Such as the polynucleotide of claim 34 or 35, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 41. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 12. For example, the polynucleotide of claim 37, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 27. Such as the polynucleotide of claim 37 or 38, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 42. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 13. For example, the polynucleotide of claim 40, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 28. Such as the polynucleotide of claim 40 or 41, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 43. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 14. Such as the polynucleotide of claim 43, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 29. Such as the polynucleotide of claim 43 or 44, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 44. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 15. Such as the polynucleotide of claim 46, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 30. Such as the polynucleotide of claim 46 or 47, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 45. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 226. Such as the polynucleotide of claim 49, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 230. Such as the polynucleotide of claim 49 or 50, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 234. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 227. Such as the polynucleotide of claim 52, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 231. Such as the polynucleotide of claim 52 or 53, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 235. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 228. Such as the polynucleotide of claim 55, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 232. Such as the polynucleotide of claim 55 or 56, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 236. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 229. Such as the polynucleotide of claim 58, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 233. Such as the polynucleotide of claim 58 or 59, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 237. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 238. Such as the polynucleotide of claim 61, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 242. Such as the polynucleotide of claim 61 or 62, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 246. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 239. For example, the polynucleotide of claim 64, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 243. Such as the polynucleotide of claim 64 or 65, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 247. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 240. Such as the polynucleotide of claim 67, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 244. Such as the polynucleotide of claim 67 or 68, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 248. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 241. Such as the polynucleotide of claim 70, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 245. Such as the polynucleotide of claim 70 or 71, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 249. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 47. Such as the polynucleotide of claim 73, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 64. Such as the polynucleotide of claim 73 or 74, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 81. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 48. Such as the polynucleotide of claim 76, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 65. Such as the polynucleotide of claim 76 or 77, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 82. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 49. Such as the polynucleotide of claim 79, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 66. Such as the polynucleotide of claim 79 or 80, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 83. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 50. Such as the polynucleotide of claim 82, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 67. Such as the polynucleotide of claim 82 or 83, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 84. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 51. Such as the polynucleotide of claim 85, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 68. Such as the polynucleotide of claim 85 or 86, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 85. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 52. Such as the polynucleotide of claim 88, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 69. Such as the polynucleotide of claim 88 or 89, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 86. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 53. Such as the polynucleotide of claim 91, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 70. Such as the polynucleotide of claim 91 or 92, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 87. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 54. Such as the polynucleotide of claim 94, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 71. Such as the polynucleotide of claim 94 or 95, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 88. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 55. Such as the polynucleotide of claim 97, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 72. Such as the polynucleotide of claim 97 or 98, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 89. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 56. For example, the polynucleotide of claim 100, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 73. For example, the polynucleotide of claim 100 or 101, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 90. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 57. Such as the polynucleotide of claim 103, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 74. Such as the polynucleotide of claim 103 or 104, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 91. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 58. Such as the polynucleotide of claim 106, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 75. Such as the polynucleotide of claim 106 or 107, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 92. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 59. Such as the polynucleotide of claim 109, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 76. Such as the polynucleotide of claim 109 or 110, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 93. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 60. Such as the polynucleotide of claim 112, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 77. Such as the polynucleotide of claim 112 or 113, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 94. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 61. Such as the polynucleotide of claim 115, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 78. Such as the polynucleotide of claim 115 or 116, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 95. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 62. Such as the polynucleotide of claim 118, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 79. Such as the polynucleotide of claim 118 or 119, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 96. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 98. For example, the polynucleotide of claim 121, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 110. Such as the polynucleotide of claim 121 or 122, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 122. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 99. For example, the polynucleotide of claim 124, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 111. Such as the polynucleotide of claim 124 or 125, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 123. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 100. For example, the polynucleotide of claim 127, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 112. Such as the polynucleotide of claim 127 or 128, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 124. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 101. For example, the polynucleotide of claim 130, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 113. Such as the polynucleotide of claim 130 or 131, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 125. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 102. Such as the polynucleotide of claim 133, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 114. Such as the polynucleotide of claim 133 or 134, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 126. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 103. Such as the polynucleotide of claim 136, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 115. Such as the polynucleotide of claim 136 or 137, wherein the follower strand sequence has the following nucleic acid sequence: SEQ ID NO: 127. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 104. Such as the polynucleotide of claim 139, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 116. Such as the polynucleotide of claim 139 or 140, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 128. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 105. For example, the polynucleotide of claim 142, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 117. Such as the polynucleotide of claim 142 or 143, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 129. The polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 106. For example, the polynucleotide of claim 145, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 118. Such as the polynucleotide of claim 145 or 146, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 130. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 107. For example, the polynucleotide of claim 148, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 119. Such as the polynucleotide of claim 148 or 149, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 131. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 108. Such as the polynucleotide of claim 151, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 120. Such as the polynucleotide of claim 151 or 152, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 132. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 134. For example, the polynucleotide of claim 154, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 140. Such as the polynucleotide of claim 154 or 155, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 146. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 135. For example, the polynucleotide of claim 157, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 141. Such as the polynucleotide of claim 157 or 158, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 147. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 136. For example, the polynucleotide of claim 160, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 142. Such as the polynucleotide of claim 160 or 161, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 148. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 137. For example, the polynucleotide of claim 163, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 143. Such as the polynucleotide of claim 163 or 164, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 149. Such as the polynucleotide of any one of claims 1 to 6, wherein the stem-loop region is a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 138. For example, the polynucleotide of claim 166, wherein the guide sequence has the following nucleic acid sequence: SEQ ID NO: 144. Such as the polynucleotide of claim 166 or 167, wherein the follower sequence has the following nucleic acid sequence: SEQ ID NO: 150. The polynucleotide of any one of claims 1 to 168, wherein the polynucleotide comprises an antisense oligonucleotide (ASO). Such as the polynucleotide of any one of claims 1 to 169, wherein the polynucleotide contains short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) or short hairpin-applicable miRNA (short hairpin-adapted miRNA, shmiRNA). The polynucleotide of any one of claims 1 to 170, wherein the polynucleotide is between 19 and 21 nucleotides. Such as the polynucleotide of claim 171, wherein the polynucleotide is 19 nucleotides. Such as the polynucleotide of claim 172, wherein the polynucleotide is 20 nucleotides. Such as the polynucleotide of claim 173, wherein the polynucleotide is 21 nucleotides. Such as the polynucleotide of any one of claims 1 to 174, wherein the Grik2 mRNA is encoded by the following nucleic acid sequences: 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. The polynucleotide of any one of claims 1 to 175, wherein the polynucleotide is capable of reducing the level of GluK2 protein in cells. Such as the polynucleotide of claim 176, wherein 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%. Such as the polynucleotide of claim 176 or 177, wherein the cell is a human cell. The polynucleotide of any one of claims 176 to 178, wherein the cell is a neuron. The polynucleotide of claim 179, wherein the neuron is a hippocampal neuron. The polynucleotide of claim 180, wherein the hippocampal neuron is a dentate granule cell (DGC) or a glutamatergic pyramidal neuron. A vector comprising the polynucleotide of any one of claims 1 to 181. A vector such as claim 182, wherein the vector is replication-defective. Such as the vector of claim 182 or 183, wherein the vector is a mammalian, insect, bacterial or viral vector. Such as the vehicle of any one of claims 182 to 184, wherein the vehicle is an expression vehicle. Such as the vector of claim 184 or 185, wherein the viral vector system is selected from the group consisting of: adeno-associated virus (AAV), retrovirus, adenovirus, parvovirus, coronavirus, negative-stranded RNA virus , orthomyxovirus, rhabdovirus, paramyxovirus, orthostranded RNA virus, picornavirus, alphavirus, double-stranded DNA virus, herpes virus, Epstein-Barr Viruses, cytomegalovirus, fowlpox virus and canarypox virus. A vector such as claim 186, wherein the vector is an AAV vector. Such as the vector of claim 187, wherein the AAV vector is an AAV5, AAV9 or AAVrh10 vector. A manifestation cassette comprising a polynucleotide containing a stem-loop sequence having at least 85% sequence identity to the nucleic acid sequence of any of the following: SEQ ID NOs: 1 to 15, 46 to 62, 97 to 108, 133 to 138, 226 to 229, and 238 to 241, such as polynucleotides containing at least 85% sequence identity to the nucleic acid sequences of any of the following: SEQ ID NOs: 4, 135, and 256 to 261. Such as the performance cassette of claim 189, wherein the performance cassette includes a 5' flanking area, a ring area and a 3' flanking area. The expression cassette of claim 190, wherein the 5' flanking region comprises a polynucleotide having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NO: 217, 220, or 223. The expression cassette of claim 190 or 191, wherein the 3' flanking region comprises a polynucleotide having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NO: 218, 221, or 224. A cassette as claimed in any one of claims 190 to 192, wherein the 5' flanking region includes a 5' spacer sequence and a 5' flanking sequence. A cassette as claimed in any one of claims 190 to 193, wherein the 3' flanking region includes a 3' spacer sequence and a 3' flanking sequence. A cassette as expressed in any one of claims 190 to 194, wherein the loop region includes a microRNA loop sequence that is an E-miR-30, miR-218-1 or E-miR-124-3 sequence. The expression cassette of claim 195, wherein the microRNA loop sequence comprises a polynucleotide having at least 85% sequence identity with any of the following nucleic acid sequences: SEQ ID NO: 219, 222, or 225. The performance cassette of any one of claims 190 to 196, wherein the performance cassette includes a synaptophysin (hSyn) promoter or a calcium/calcin-dependent protein kinase II (CaMKII) promoter. A performance cassette that from 5' to 3' contains: (a) First promoter sequence; (b) A polynucleotide comprising a stem-loop sequence having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NOs: 1 to 19, 34 to 62, 97 to 108, 133 to 147 , 226 to 229 or 238 to 241; (c) optionally, the second promoter sequence; (d) A polynucleotide comprising a stem-loop sequence having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NOs: 1 to 19, 34 to 62, 97 to 108, 133 to 147 , 226 to 229 or 238 to 241. The expression cassette of claim 198, wherein the polynucleotide comprising the stem-loop sequence having any of the following nucleic acid sequences includes a follower sequence that is complementary or substantially complementary to the leader sequence: SEQ ID NOs: 1 to 19, 34 to 62, 97 to 108, 133 to 147, 226 to 229, or 238 to 241, where the follower sequence is located 5' or 3' relative to the leader sequence. The expression cassette of claim 198 or 199, wherein the polynucleotide comprising the stem-loop sequence having any of the following nucleic acid sequences includes a 5' flanking region located 5' relative to the leader sequence: SEQ ID NO: 1 to 19, 34 to 62, 97 to 108, 133 to 147, 226 to 229 or 238 to 241. The expression cassette of any one of claims 198 to 200, wherein the polynucleotide comprising a stem-loop sequence having a nucleic acid sequence of any of the following comprises a 3' flanking region located 3' relative to the leader sequence :SEQ ID NO: 1 to 19, 34 to 62, 97 to 108, 133 to 147, 226 to 229 or 238 to 241. The expression cassette of any one of claims 198 to 201, wherein the polynucleotide comprising a stem-loop sequence having a nucleic acid sequence of any of the following includes a loop region between the leader sequence and the follower sequence: SEQ ID NO: 1 to 19, 34 to 62, 97 to 108, 133 to 147, 226 to 229 or 238 to 241, wherein the loop region contains a microRNA loop sequence. The performance cassette of any one of claims 198 to 202, wherein the first promoter and/or optionally the second promoter is selected from the group consisting of: an hSyn promoter or a CaMKII promoter. The expression cassette of any one of claims 198 to 203, wherein the 5' flanking region comprises a polynucleotide having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NO: 217, 220 or 223. The expression cassette of any one of claims 198 to 204, wherein the 3' flanking region comprises a polynucleotide having at least 85% sequence identity with the nucleic acid sequence of any of the following: SEQ ID NO: 218, 221 or 224. Such as the expression cassette of any one of claims 198 to 205, wherein the microRNA loop sequence is an E-miR-30, miR-218-1 or E-miR-124-3 sequence. The expression cassette of claim 206, wherein the microRNA loop sequence comprises a polynucleotide having at least 85% sequence identity with any of the following nucleic acid sequences: SEQ ID NO: 219, 222, or 225. The performance cassette of any one of claims 198 to 207, wherein the performance cassette contains a 5'-inverted terminal repeat (ITR) sequence at the 5' end of the performance cassette and a 3' end of the performance cassette 3'-ITR sequence at the end. Such as the representation cassette of claim 208, wherein the 5'-ITR sequence and the 3' ITR sequence are AAV2 5'-ITR sequence and 3' ITR sequence. The expression cassette of claim 208 or 209, wherein the 5'-ITR sequence comprises a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 208 or SEQ ID NO: 209. The expression cassette of any one of claims 208 to 210, wherein the 3'-ITR sequence comprises a polynucleotide having at least 85% sequence identity with any of the following nucleic acid sequences: SEQ ID NO: 210 to 212. A cassette as claimed in any one of claims 198 to 211, further comprising an enhancer sequence. The expression cassette of claim 212, wherein the enhancer sequence comprises a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 207. A cassette as claimed in any one of claims 198 to 213, further comprising an intron sequence. A cassette as expressed in claim 214, wherein the intron sequence comprises a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 205 or SEQ ID NO: 206. The performance cassette of any one of claims 198 to 215 further includes one or more polyadenylation signal sequences. Such as the performance cassette of claim 216, wherein the one or more polyadenylation sequences are a rabbit β-globin (RBG) polyadenylation sequence or a bovine growth hormone (BGH) polyadenylation sequence. Such as the expression cassette of claim 217, wherein the RBG polyadenylation message sequence comprises a polynucleotide having at least 85% sequence identity with any of the following nucleic acid sequences: SEQ ID NOs: 213 to 215. Such as the expression cassette of claim 217, wherein the BGH polyadenylation message sequence includes a polynucleotide having at least 85% sequence identity with the following nucleic acid sequence: SEQ ID NO: 216. A representation cassette according to any one of claims 198 to 219, wherein the representation cassette is incorporated into a carrier according to any one of claims 182 to 188. A performance cassette as claimed in any one of requests 198 to 220, wherein the performance cassette contains at least 80% sequence identity to the following sequence: SEQ ID NO: 256. For example, the performance cassette of any one of request items 198 to 221, wherein the performance cassette contains at least 85%, 90%, 95%, or 97% sequence identity with the following sequence: SEQ ID NO: 256. A method for inhibiting the expression of Grik2 in a cell, which comprises coordinating the cell with at least one polynucleotide as in any one of claims 1 to 181, a vector as in any one of claims 182 to 188, or a vector as in claim 189 to 181 Any one of 222 indicates cassette contact. The method of claim 223, wherein the polynucleotide specifically hybridizes to Grik2 mRNA and inhibits or reduces the expression of Grik2 in the cell. The method of claim 223 or 224, wherein the method reduces the level of GluK2 protein in the cell. The method of claim 225, wherein 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%. The method of any one of claims 223 to 226, wherein the cells are human cells. The method of any one of claims 223 to 227, wherein the cell is a neuron. The method of claim 228, wherein the neuron is a hippocampal neuron. The method of claim 229, wherein the hippocampal neuron is a DGC or glutamatergic pyramidal neuron. The method of claim 230, wherein the DGC contains aberrant recurrent mossy fiber axon. A method of treating or improving a condition in an individual in need thereof, comprising administering to the individual at least one polynucleotide according to any one of claims 1 to 181, or a vector according to any one of claims 182 to 188 Or a performance cassette as in any of requests 189 to 222. A composition comprising a polynucleotide as in any one of claims 1 to 181, a vector as in any one of claims 182 to 188, or an expression cassette as in any one of claims 189 to 222, for In methods of treating or ameliorating a disease or condition in an individual in need thereof. The method of claim 232 or 233, wherein the condition is epilepsy. The method of claim 234, wherein the epilepsy is temporal lobe epilepsy (TLE), chronic epilepsy, and/or refractory epilepsy. The method of claim 235, wherein the epilepsy is TLE. The method of request 236, wherein the TLE is a lateral TLE (lTLE). The method of request 236, wherein the TLE is a mesial TLE (mTLE). The method of any one of claims 233 to 238, wherein the individual is a human. A pharmaceutical composition comprising a polynucleotide as in any one of claims 1 to 169, a vector as in any one of claims 182 to 188, or a performance cartridge as in any one of claims 189 to 222, and pharmaceutically acceptable carriers, diluents or excipients. A package including a pharmaceutical composition as claimed in claim 240 and a package insert. Such as the set of claim 241, wherein the drug package insert contains instructions for the use of the pharmaceutical composition in any one of the methods of claims 232 to 239.