Compositions and methods containing nuclear small RNA (snRNA) for treating predisposing epilepsy

Abstract

An snRNA system targeting the SCN1A or SYNGAP1 RNA sequence is disclosed herein. Furthermore, a method for treating predisposing epilepsy, such as Dravet syndrome or SYNGAP1-associated epilepsy, is disclosed. TIFF2026536590000070.tif53170

Description

Technical Field

[0001] Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 592,405 filed on October 23, 2023 and U.S. Provisional Patent Application No. 63 / 592,727 filed on October 24, 2023, the contents of each of which are incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (LOCN_026_004WO_SeqList_ST26.xml; Size: 492,530 bytes; and Creation Date: October 22, 2024) is incorporated herein by reference in its entirety.

[0003] Field of the Present Disclosure The present disclosure is directed to molecular biology, gene therapy, and compositions and methods for modifying the expression and activity of RNA molecules.

Background Art

[0004] Background Nuclear small RNAs (snRNAs) are among the smallest types of RNA, with an average size of approximately 150 nucleotides. snRNAs are functional non-coding RNAs. Eukaryotic genomes encode a variety of non-coding RNAs, including snRNAs. A class of RNA that is abundant, snRNAs are localized in the nucleus and play crucial roles in intron splicing and RNA processing. During the pre-mRNA splicing process, snRNAs can form ribonucleoprotein particles (snRNPs) with other proteins. These snRNPs, along with further proteins, form large particulate complexes (spliceosomes) that bind to pre-mRNA transcripts that have not yet been spliced. In addition to splicing, snRNAs function in the maturation of nascent transcripts in the nucleus, regulate gene expression, act as splice donors in non-canonical systems, and play a role in replication-dependent 3' end processing of histone mRNA. While other protein-based gene therapy approaches carry an immunogenicity risk, U7 snRNAs can be programmed to bind to and regulate mRNA without expressing exogenous proteins, ultimately reducing this risk. Furthermore, the small size of these programmed snRNAs creates opportunities for the development of single-vector, highly specific (allele-specific), single-target, and multi-targeting gene therapy approaches.

[0005] Predisposing epilepsy is a class of seizure disorders that are caused by genetic factors, such as inherited or accidental mutations in genes.

[0006] Dravet syndrome is a rare developmental epileptic encephalopathy that typically manifests within the first year of life. Symptoms of Dravet syndrome include frequent and prolonged seizures, intellectual disability, developmental delay, behavioral disturbances, and ataxia. Individuals with Dravet syndrome also have a high risk of sudden unexpected death (SUDEP) in epilepsy. The average age of onset for SUDEP is 4.6 years.

[0007] Approximately 80% of patients with Dravet syndrome have a de novo heterozygous missense mutation or shortening in the SCN1A gene. SCN1A encodes the α subunit of voltage-gated sodium ion channel type 1 (Nav1.1), which is important for normal neuronal function. Nav1.1 is the primary Na in GABAergic inhibitory interneurons. + Nav1.1 is a channel that, in the neuron, induces rapid depolarization of the neuronal membrane during the initiation of an action potential. Mutations in Nav1.1 cause haploinsufficiency of the Nav1.1 protein. The SCN1A gene is too large for gene replacement therapy because it has 26 exons spanning 6030 bp.

[0008] The prevalence of Dravet syndrome in the United States is estimated to be approximately 1 in 15,700. Current treatments include anticonvulsant therapy that partially manages seizures but does not improve other comorbidities, ketogenic diet therapy to reduce seizure frequency, and supportive care to address neurodevelopmental challenges. Thus, there is an unmet need for new therapeutic approaches to Dravet syndrome.

[0009] SYNGAP1-associated epilepsy is a rare, hereditary disorder caused by variants in the SYNGAP1 gene. Many people develop epilepsy of varying severity, as well as / or autism spectrum disorder, and other behavioral, social, and sensory problems.

[0010] The SYNGAP1 gene is located on chromosome 6 and is responsible for producing a protein called synaptic Ras GTPase-activating protein 1 (SYNGAP1). SYNGAP1 is present at high levels in the brain and is essential for proper function and development. SYNGAP1 is a key regulator of the neural mechanisms of learning and memory at the synapses where neurons connect with each other. Types of seizures associated with SYNGAP1 include: typical and atypical absence seizures, myoclonic seizures, cataplexy, eyelid myoclonus with absence (EMA), and myoclonic cataplexy (MAE or Dose syndrome). More often, people exhibit a combination of EMA and MAE. A distinctive feature in people with this gene is eyelid myoclonus, which progresses to myoclonic cataplexy or cataplexy.

[0011] Seizures often begin between 4 months and 7 years of age, with an average age of 2 years. During the active phase of epilepsy, seizure triggers occur frequently, and in some individuals, seizures can reach more than 100 times a day. Almost all individuals affected by SYNGAP1 have developmental delay or intellectual disability. Typically, developmental delay becomes apparent during the first two years of life, which can lead to a diagnosis of intellectual disability. Two-thirds of children also exhibit hypotonia (low muscle tone), and a smaller group has ataxia (balance disorder) and / or other gait abnormalities. The incidence of SYNGAP1 is estimated to be 1 to 4 per 10,000 people. Thus, there is an unmet need for new therapeutic approaches for SYNGAP1-associated epilepsy.

[0012] In some individuals with SYNGAP1-associated epilepsy, the harmful exon 11 of SYNGAP1 contains an immature stop codon that leads to a nonsense mutation-dependent degradation mechanism, transcriptional failure, and haploinsufficiency, causing patients to experience symptoms associated with SYNGAP1-associated epilepsy. [Overview of the project]

[0013] overview A cRNA-targeting nucleic acid molecule comprising nuclear small RNA (snRNA), wherein the snRNA contains a targeting sequence that binds to an SCN1A or SYNGAP1 RNA sequence.

[0014] In some embodiments, the SCN1A RNA sequence is exon 20N. In some embodiments, the SYNGAP1 RNA sequence is exon 11. In some embodiments, the SCN1A targeting sequence includes a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 16-83. In some embodiments, the SYNGAP1 targeting sequence includes a nucleic acid sequence shown in any one of SEQ ID NO: 181-279.

[0015] In some embodiments, the snRNA includes a stem-loop (SL). In some embodiments, the SL includes one or more nucleic acid sequences represented by any one of SEQ ID NO: 84-124. In some embodiments, the SL includes one or more nucleic acid sequences that are independently at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to the nucleic acid sequences represented by any one of SEQ ID NO: 84-124. In some embodiments, the SL includes or consists of nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to the nucleic acid sequence represented by SEQ ID NO: 97.

[0016] This disclosure provides an RNA-targeting nucleic acid molecule comprising nuclear small RNA (snRNA), wherein the snRNA comprises a targeting sequence that binds to an SCN1A RNA sequence and an SL containing the nucleic acid sequence shown in SEQ ID NO: 97, where the targeting sequence is located at the 5' end of the SL.

[0017] In some embodiments, the SCN1A RNA sequence is exon 20N. In some embodiments, the SCN1A targeting sequence includes a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 16-83.

[0018] In some aspects, the SCN1A RNA sequence is either a premRNA sequence or an mRNA sequence.

[0019] In some embodiments, the snRNA includes an Sm-binding domain (SmBD). In some embodiments, the SmBD is U1, U2, U4, or U5 SmBD. In some embodiments, the SmBD includes a nucleic acid sequence represented by either SEQ ID NO: 160 or 161.

[0020] In some embodiments, the snRNA includes a 5' interaction stabilization domain (5'ISD). In some embodiments, the 5'ISD includes the nucleotide sequence ggagt, cctct, ggaggt, cctcct, agccag, ggaag, gaagaag, gttg, ccgaa, taaggag, gaag, or ggctt.

[0021] This disclosure provides a vector comprising one or more snRNAs as in any aspect of this disclosure. In some aspects, the vector is an adeno-associated virus (AAV) vector.

[0022] In some embodiments, the snRNA is operably linked to a promoter. In some embodiments, the snRNA is operably linked to a U7 promoter or a U1 promoter.

[0023] In some embodiments, the snRNA is operably linked to a downstream terminator (DT). In some embodiments, the snRNA is operably linked to the downstream terminator of U7 or the downstream terminator of U1.

[0024] In some embodiments, the vector comprises at least 1, at least 2, at least 3, at least 4, or at least 5 snRNAs. In some embodiments, each of the at least 1, at least 2, at least 3, at least 4, or at least 5 snRNAs targets the same target RNA sequence.

[0025] In some embodiments, each snRNA is separated by a buffer sequence. In some embodiments, the buffer sequence comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 151 to 157.

[0026] In some embodiments, the vector comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 162 to 180.

[0027] The present disclosure provides a method of targeting one or more target RNAs of interest to effect exon skipping of the one or more target RNAs, which method comprises the step of contacting the snRNA of any embodiment of the present disclosure with a cell comprising the one or more target RNAs.

[0028] This disclosure provides an SCN1A RNA targeting nucleic acid molecule containing a targeting sequence represented by one of SEQ ID NO: 16-83. This disclosure also provides a SYNGAP1 RNA targeting nucleic acid molecule containing a targeting sequence represented by one of SEQ ID NO: 181-279.

[0029] This disclosure provides a polynucleotide or vector comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to a nucleic acid sequence represented by any one of SEQ ID NO: 162-180.

[0030] This disclosure provides recombinant AAV (rAAV) comprising: (a) an AAV capsid comprising an AAV capsid protein; and (b) a vector genome comprising an snRNA or RNA-targeting nucleic acid molecule as disclosed herein.

[0031] In some embodiments, the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence and a 3' ITR. In some embodiments, the vector genome comprises a 5' ITR sequence, an snRNA or RNA-targeting nucleic acid molecule, and a 3' ITR sequence in a 5'-to-3' direction.

[0032] This disclosure provides recombinant AAV (rAAV) comprising: an AAV capsid containing an AAV capsid protein; and a vector genome comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 162–180.

[0033] In some embodiments, the AAV capsid comprises an AAV capsid protein of an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and their variants.

[0034] In some embodiments, the vector genome is single-stranded or self-complementary.

[0035] In some embodiments, rAAV is uncopyable.

[0036] This disclosure provides pharmaceutical compositions comprising snRNA, RNA-targeting nucleic acid molecules, vectors, polynucleotides, or rAAVs as disclosed herein.

[0037] A method for exon skipping one or more target RNAs of interest, comprising the step of contacting an snRNA, RNA targeting nucleic acid molecule, vector, polynucleotide, or rAAV disclosed herein with a cell containing the one or more target RNAs.

[0038] In some embodiments, one or more target RNAs of interest contain the harmful exon 20N of the human sodium channel Nav1.1 gene.

[0039] This disclosure provides a method for treating a disease or disorder in a subject, the method comprising the step of administering to the subject an snRNA, RNA-targeting nucleic acid molecule, vector, polynucleotide, rAAV, or pharmaceutical composition disclosed herein.

[0040] In some embodiments, the disease or disorder is predisposing epilepsy. In some embodiments, the disease or disorder is Dravet syndrome. In some embodiments, the disease or disorder is SYNGAP1-associated epilepsy.

[0041] In some embodiments, administration is systemic, intravenous, or intraventricular.

[0042] This disclosure provides the use of the snRNA or RNA targeting nucleic acid molecules disclosed herein for targeting one or more target RNAs of interest and performing exon skipping of said one or more target RNAs.

[0043] In some embodiments, one or more target RNAs are present in the cell.

[0044] This disclosure provides the use of the snRNAs, RNA-targeting nucleic acid molecules, vectors, polynucleotides, rAAVs, or pharmaceutical compositions disclosed herein for treating diseases or disorders in subjects.

[0045] In some embodiments, the disease or disorder is predisposed epilepsy. In some embodiments, the disease or disorder is Dravet syndrome.

[0046] In some embodiments, administration is systemic, intravenous, or intraventricular. [Brief explanation of the drawing]

[0047] [Figure 1A] This paper describes the mechanism of skipping of SCN1A harmful exon 20N by U7 snRNA to restore the frame and function of the sodium channel Nav1.1 in Dravet syndrome. U7 snRNA binds to splicing regulatory sequences (acceptor and donor sites, or splicing enhancer sequences) within SCN1A harmful exon 20N, promoting skipping and the restoration of frame and function. PTC stands for immature stop codon; NMD stands for nonsense mutation-dependent degradation mechanism. [Figure 1B]The images show tapestation images of RT-PCR products after treatment with U7 snRNA using a single U7 spacer (in pcDNA), indicating bands where SCN1A exon 20N is not skipped (containing the harmful exon, upper fragment) or products where it is skipped (excluding the harmful exon, lower band). Non-targeting U7 (NT), empty pcDNA plasmid (pc), and untreated cells (UNT) were used as negative controls. [Figure 1C] Figure 1B shows the quantification of the gel, plotted as the percentage of harmful exon 20N inclusion after treatment with multiple U7 snRNA single spacers in Hela cells. The dashed line represents the average harmful exon 20N inclusion under control conditions, either as untreated cells or as cells transfected with empty plasmids or non-targeting (NT) U7 snRNAs. [Figure 2A] The images show tapestations of RT-PCR products after treatment with U7 snRNA using a single and fused U7 spacer (cloned within a pcDNA plasmid). Bands where SCN1A exon 20N is not skipped (containing the harmful exon, upper fragment) or products where it is skipped (excluding the harmful exon, lower band) are shown. Untargeted U7 (NT) and untreated cells (UNT) were used as negative controls. [Figure 2B] Figure 2A shows the quantification of the gel, plotted as the percentage of harmful exon 20N inclusion after treatment with multiple U7 snRNA spacers in HepG2 cells. [Figure 2C] This shows the expression of productive (harmless exon 20N) SCN1A, as determined by qRT-PCR after treatment with single and fusion U7 snRNAs. [Figure 3A]This study quantifies the percentage of harmful exon 20N inclusion in RT-PCR products after treatment with U7 snRNA using z15 / z6 and z11 / z15, which are fusion synthetic U7 spacers with further modifications to the ISD and stem-loop. Untargeted U7 (NT) and untreated cells (UNT) were used as negative controls. [Figure 3B] This shows the expression of productive (harmless exon 20N) SCN1A, as determined by qRT-PCR after treatment with U7 snRNA. [Figure 4A] The image shows tapestation of RT-PCR products to detect exon 20N skipping after treatment with U7 snRNA using AAV9 (a dual U7 snRNA cassette expressing a fusion spacer under the mouse U7 promoter and mouse U1 promoter) while increasing MOI. The gel image shows SCN1A harmful exon 20N (unskipped band, top) and skipped product (bottom band). Cells with AAV9 null (empty capsid) and untreated (UNT) cells were used as negative controls. [Figure 4B] Figure 4A shows the quantification of the gels shown, plotted as the percentage of harmful exon 20N inclusion after transduction in AAV9 using two U7 snRNA vectors, A05290 and A05291. [Figure 4C] This shows the quantitative level of productive (harmless exon-free) SCN1A RNA 7 days after transduction using AAV9 containing a dual U7 snRNA cassette in SCN1A + / - GABAergic neurons. [Figure 5A] This diagram shows a schematic of the study design for an in vivo study using wild-type (WT) mice. The mice were treated with scAAV9-U7 snRNA targeting the SCN1A harmful exon, which was delivered intraventricularly (ICV) at P0, and collected at P20. [Figure 5B]Representative tapestation images of RT-PCR products from cortical and hippocampal samples after treatment with U7 snRNA using two read constructs, A05290 and A05291, are shown. The images indicate bands where the SCN1A harmful exon 21N (the mouse equivalent of the harmful exon 20N in humans) is not skipped (upper fragment), or products where it is skipped (lower band). Vehicle-treated samples were used as negative controls. [Figure 5C] Figure 5B shows the quantification of gels, along with samples from further cortical sections (1, 2, and 3) and further hippocampal sections (1 and 2), plotted as the percentage of harmful exon 21N inclusion after transduction in AAV9 using two different U7 snRNA vectors. [Figure 5D] This shows the quantification of productive (i.e., free of harmful exons) SCN1A mRNA levels in three cortical areas and two hippocampal areas 20 days after ICV injection of AAV9 expressing a dual U7 snRNA cassette. [Figure 5E] This shows the expression levels of the SCN1A / Nav1.1 protein in the cortex (CTX) and hippocampus (Hippo) 20 days after ICV injection of AAV9 expressing a dual U7 snRNA cassette. [Figure 5F] This shows snRNA expression in the cortex and hippocampus (Hipp) after treatment with AAV9, which expresses snRNA. [Figure 6A] This schematic diagram illustrates the inclusion of SYNGAP harmful exon 11 (shown as a shaded rectangle), which contains an immature stop codon (PTC) and causes transcript degradation and haploinsufficiency via a nonsense mutation-dependent degradation mechanism (NMD). [Figure 6B]This is a schematic diagram illustrating the mechanism of action of U7 snRNA-mediated skipping of SYNGAP harmful exon 11 to restore frame and function. U7 snRNA was engineered to bind to a splicing regulatory sequence within SYNGAP harmful exon 11, thereby promoting skipping and the restoration of frame and function. [Figure 6C] These are tapestation images of RT-PCR products after treatment with U7 snRNA using individual U7 spacers (in pcDNA, z22, z23, and z24), showing bands where SYNGAP harmful exon 11 is not skipped (those containing the harmful exon, upper fragment) or products where it is skipped (those with the harmful exon excluded, lower band). Non-targeting U7(NT) was used as a negative control. ASO, which has been previously shown to promote the exclusion of harmful exon 11, was used as a positive control. [Figure 6D] This graph quantifies tapestation results in Hek293-T cells, plotting the percentage of harmful exon elimination after treatment with U7 snRNA, which is a single and fusion spacer targeting distinct areas of the SYNGAP1 premRNA. [Figure 7-1]Figure 7A is a graph showing the quantification of endogenous SYNGAP1 harmful exon 11 skipping as a percentage, determined by splicing assay after treatment with monosynthetic U7 snRNA in HEK293-T cells. Figure 7B is a graph showing the quantification of productive SYNGAP1 mRNA expression as determined by qRT-PCR after treatment with monosynthetic U7 snRNA in HEK293-T cells. Non-targeting U7(NT) was used as a negative control. Figure 7C is a graph showing the quantification of endogenous SYNGAP1 harmful exon 11 skipping as a percentage, determined by splicing assay after treatment with novel optimized monosynthetic and fusion U7 snRNA in HEK293-T cells. Figure 7D is a graph showing the quantification of productive SYNGAP1 mRNA expression as determined by qRT-PCR after treatment with novel optimized monosynthetic and fusion U7 snRNA in HEK293-T cells. Non-targeting U7(NT) was used as a negative control. [Figure 7-2] Please refer to the explanation in Figure 7-1. [Figure 8A] This is a tapestation image of RT-PCR products as levels of SYNGAP1 exon 11 in neural progenitor cells (NPCs) of a patient containing the R1240X mutation. The patient's NPCs show high levels of harmful SYNGAP1 exon 11 (upper band). [Figure 8B] This graph shows the quantification of RT-PCR products as the percentage of skipping of harmful exon 11 of SYNGAP1 after treatment with U7 snRNA in patient-derived NPCs. Non-targeting U7(NT) was used as a negative control. [Figure 8C] This graph shows the quantification of productive (harmless exon 11-free) SYNGAP1 mRNA expression, determined by qRT-PCR after treatment with U7 snRNA. [Modes for carrying out the invention]

[0048] Detailed explanation This disclosure provides a gene therapy composition comprising a therapeutic RNA targeting platform, the platform comprising nuclear small RNA (snRNA) that targets a precursor mRNA (premRNA) sequence or mRNA sequence encoding Nav1.1 (SCN1A). The targeted premRNA sequence or mRNA sequence may include the exon region of SCN1A and / or the splicing regulatory sequence of SCN1A.

[0049] This disclosure provides a gene therapy composition comprising a therapeutic RNA targeting platform, the platform comprising nuclear small RNA (snRNA) that targets a precursor mRNA (premRNA) sequence or mRNA sequence encoding synaptic Ras GTPase-activating protein 1 (SYNGAP1). The targeted premRNA sequence or mRNA sequence may include the exon region of SYNGAP1 and / or the splicing regulatory sequence of SYNGAP1.

[0050] This specification discloses compositions comprising nucleic acid molecules and vectors comprising one or more snRNA constructs targeting SCN1A. The snRNA molecules of this disclosure may be non-natural, modified, and / or engineered snRNAs (esnRNAs). In some embodiments, the snRNAs or esnRNAs of this disclosure targeting SCN1A include a mutant snRNA stem-loop. In some embodiments, the snRNAs of this disclosure targeting SCN1A include a natural stem-loop.

[0051] This specification discloses compositions comprising nucleic acid molecules and vectors comprising one or more snRNA constructs targeting SYNGAP1. The snRNA molecules of this disclosure may be non-natural, modified, and / or engineered snRNAs (esnRNAs). In some embodiments, the snRNAs or esnRNAs of this disclosure targeting SYNGAP1 include a mutant snRNA stem-loop. In some embodiments, the snRNAs of this disclosure targeting SYNGAP1 include a natural stem-loop.

[0052] Nuclear small ribonucleic acid (snRNA) is an essential component of nuclear small ribonucleoprotein complexes (snRNPs). When assembled with other proteins, it forms a large ribonucleoprotein complex known as the spliceosome, an intracellular mechanism that mediates the entire mRNA maturation process. The spliceosome plays a role in precursor mRNA splicing, the process of removing introns from RNA transcripts before protein production. Individual snRNAs are generally about 250 nucleotides or less in size. For example, U1 snRNA is 164 nucleotides long and is encoded by a gene that exists in several copies within the human genome. U1 snRNA corresponds to the ribonucleic acid component of the nuclear particle U1 snRNP. U1 snRNAs have a three-dimensional stem-loop structure and generally contain a single-stranded sequence of approximately 9 nucleotides in length within their 5' region, which can bind to a splicing donor site on the premRNA molecule through complementary base pairing (Horowitz et al., 1994, Trends Genet., 10(3):100-6). The various snRNAs of the spliceosome are designated as U1, U2, U4, U5, U6, U4ATAC, U6ATAC, U7, U11, and U12, due to the high amount of uridylic acid they contain (Mattaj et al., 1993, FASEB J, 15, 7:47-53).

[0053] snRNA systems can be used to address harmful mutations. For example, antisense oligonucleotides that interfere with splice sites and regulatory elements within exons containing harmful mutations can induce specific exon skipping at the preRNA level. Such antisense sequences can be packaged within snRNA sequences delivered using viral vectors, which carry nucleic acid sequences capable of transcribing the snRNA. While U7 snRNA is intrinsically involved in the 3' end processing of histone premRNA, small changes in its binding site to Sm / Lsm proteins can transform it into a versatile tool for splicing regulation.

[0054] Most U-rich snRNPs are complexes that mediate premRNA splicing. U7 snRNPs are an exception. While U7 is not involved in splicing, it is a crucial factor in the unique 3' end processing of replication-dependent histone mRNA. By modifying the histone-binding sequence and Sm motif of U7 snRNA, U7 can cease to be involved in histone premRNA processing and instead target premRNA or smRNA for blocking or splicing regulation. In this manner, U7 snRNA can be used as an effective gene therapy platform. The U7 snRNA platform also has the added advantage of being small in size, which allows it to accumulate in the nucleus without causing cytotoxicity and exhibits little to no immunoreactivity (Gadgil et al., 2021, J Gene Med, 23(4): e3321). The U7 snRNA platform is described in detail in International Publication No. WO 2023 / 168458, which is incorporated herein by reference in its entirety.

[0055] This specification discloses esnRNAs containing stem-loops (SLs) in several aspects. Compensatory modifications made to the native stem-loop can create SLs that communicate (fold and anneal) more effectively with the snRNA interaction stabilization domain (ISD) compared to the native stem-loop sequence, which in turn create an snRNA platform with increased stability. U7 snRNAs have been previously shown to be programmable to modulate mRNA. This specification discloses improved programmed and manipulated snRNAs that can be used as tools for gene therapy.

[0056] The snRNA systems disclosed herein are configured to bind to a targeted SCN1A or SYNGAP1 RNA sequence in order to modulate RNA splicing that can induce single or multiple exon skipping or exon inclusion in the sequence of the targeted SCN1A or SYNGAP1 RNA. SCN1A-targeting snRNAs are configured to bind to an SCN1A premRNA molecule at a site that modulates RNA splicing. SYNGAP1-targeting snRNAs are configured to bind to a SYNGAP1 premRNA molecule at a site that modulates RNA splicing. Sites that modulate splicing may include splice acceptor sequences, splice donor sequences, and exon splice enhancer sequences. The snRNA sequences of this disclosure can induce exon skipping (of single or multiple exons) of a targeted exon sequence.

[0057] Nonproductive splicing events, such as the inclusion of harmful exons containing immature stop codons, are innate regulatory mechanisms for reducing the expression of unwanted genes during development or in certain tissues. The SCN1A premRNA contains a "harmful exon" (exon 20N in humans, exon 21N in mice), which contains an immature stop codon. The SYNGAP1 premRNA contains a "harmful exon" (exon 11 in humans), which contains an immature stop codon. When such exons are transcribed, a nonsense mutation-dependent degradation mechanism of RNA is triggered. While we do not wish to be bound by theory, it is hypothesized that using the U7 snRNA platform to facilitate the skipping of the "harmful exon" (exon 20N) in the SCN1A premRNA may increase the expression of the full-length SCN1A transcript, and consequently, promote the expression of a restored, functional Nav1.1 protein. Furthermore, while we do not wish to be bound by theory, we hypothesize that by using the U7 snRNA platform to promote the skipping of the "harmful exon" (exon 11) in SYNGAP1 premRNA, the expression of the full-length SYNGAP1 transcript may be increased, and as a result, the expression of a restored, functional SYNGAP1 protein may be promoted.

[0058] In one embodiment, these snRNAs are human snRNAs. In another embodiment, these snRNAs are mouse snRNAs. In another embodiment, the snRNAs are of any species. In another embodiment, the snRNAs are a combination of human and mouse snRNAs. In one embodiment, the U7 snRNA is either human U7 snRNA or mouse U7 snRNA. In another embodiment disclosed herein, the snRNAs comprise various types of snRNAs (e.g., U1-U12) by combining domains of endogenous snRNAs, which is done for the purpose of fine-tuning platform stability and / or reducing off-target effects. For example, in one embodiment, the snRNA system comprises a combination of a human or mouse U7 snRNA component and a human or mouse U1 snRNA component.

[0059] Further operations may be performed on snRNA or esnRNA containing SL to add additional elements that can modulate the processing and abundance of RNA. In one embodiment, further operations may be performed on esnRNA at the 5' or 3' end to add additional elements that can modulate the processing, stability, and abundance of esnRNA. In another embodiment, such elements may include, but are not limited to, stem-loops, hairpins, GC clamps, kissing loops, triple helixes, quadruple helixes, and protein-binding sites.

[0060] As long as an appropriate spacer or target sequence TS is included in the design of the therapeutic composition, the snRNA platform and any part thereof can be used in therapeutic environments and situations. In one embodiment, the therapeutic snRNA composition is used to treat diseases associated with dysregulated NaV1.1, mutated NaV1.1, or non-functional NaV1.1. In some aspects, the disease or disorder is Dravet syndrome.

[0061] Targeting array An snRNA system can be programmed to include a targeting sequence (TS) (also called a “spacer”) that targets an RNA of interest. An snRNA system can be programmed to have one or more targeting sequences that target one or more RNAs of interest. In some aspects, the targeting sequence is a 5' targeting sequence (5'TS) that targets one or more RNAs of interest. In this context, 5' refers to the 5' end of the snRNA insert and not necessarily to the overall vector configuration containing one or more snRNA inserts. The TS may be located at or near the 5' end of the snRNA. In another embodiment, particularly when the snRNA construct is not a U7-based snRNA, the targeting sequence (TS) may be located at or near the 3' position of the snRNA construct, thereby creating a 3' targeting sequence (3'TS).

[0062] The targeting sequences of this disclosure, including the 5'TS and 3'TS, may be approximately 1 to approximately 200 nucleotides in length. In some aspects, the targeting sequences of this disclosure are approximately 10 to approximately 150 nucleotides in length. In some aspects, the targeting sequences of this disclosure are approximately 10 to approximately 100 nucleotides in length. In some aspects, the targeting sequences of this disclosure are approximately 20 to approximately 60 nucleotides in length. In some aspects, the targeting sequences of this disclosure are at least approximately 10, 20, 30, 40, 50, 60, or approximately 70 nucleotides in length.

[0063] The snRNA compositions of this disclosure may comprise a plurality of targeting sequences, each of which binds to a separate RNA sequence. In some aspects, the snRNAs of this disclosure comprise a fusion targeting sequence. In some aspects, the fusion targeting sequence is a nucleic acid sequence comprising two targeting sequences, which are directly linked or linked by one or more linker nucleic acid sequences, each of which binds to a different target RNA sequence.

[0064] As an example, U7 snRNAs can be programmed by replacing a histone mRNA binding sequence with a sequence complementary to the target of interest. In some aspects, the snRNA systems of this disclosure bind to a target mRNA sequence or a target pre-mRNA sequence of interest. The exemplary snRNA systems shown herein induce exon skipping (e.g., SCN1A exon 20N skipping when treating Dravet syndrome; or SYNGAP1 exon 11 skipping when treating SYNGAP1-associated epilepsy).

[0065] In some aspects, the snRNAs of this disclosure target a premRNA or mRNA sequence encoding the NaV1.1 protein. SCN1A is the gene encoding the protein NaV1.1. Mutations in SCN1A are associated with Dravet syndrome. In some aspects, the SCN1A RNA sequence targeted by the snRNA composition of this disclosure may be any SCN1A exon or SCN1A intron RNA sequence. In some aspects, the SCN1A RNA sequence targeted by the snRNA composition of this disclosure is an exon 20N or exon 21N SCN1A RNA sequence. In some aspects, the SCN1A RNA sequence targeted by the snRNA composition of this disclosure is a human exon 20N SCN1A RNA sequence. In some aspects, the SCN1A RNA sequence targeted by the snRNA composition of this disclosure is a mouse exon 21N SCN1A RNA sequence. In some embodiments, the SCN1A RNA sequence targeted by the snRNA of this disclosure is a splice acceptor sequence, a splice donor sequence, or an exon splice enhancer sequence.

[0066] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 1.

[0067] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2.

[0068] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 3.

[0069] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 4. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 4.

[0070] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 5.

[0071] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 6. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 6.

[0072] A includes or consists of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 7.

[0073] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 8. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 8.

[0074] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 9.

[0075] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 10.

[0076] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 11. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 11.

[0077] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 12.

[0078] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 13.

[0079] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 14.

[0080] In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes or consists of SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 15.

[0081] The target sequence that binds to SCN1A exon 20N may include, be essentially, or may be essentially, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences shown in Table 1 below.

[0082] (Table 1) Exon 20N targeting array (single spacer) TIFF2026536590000002.tif191161

[0083] In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 16. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 17. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 18. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 19. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 20. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 21. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 22. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 23. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 24. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 25. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 26. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 27. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 28. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 29. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 30. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 31.In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 32. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 33. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 34. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 35. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 36. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 37. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 38. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 39. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 40. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 41. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 42. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 43. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 44. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 45. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 46. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 47.In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 48. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 49. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 50. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 51.

[0084] To prepare a fusion spacer, the sequences shown in Table 1 may be combined. Any first sequence shown in Table 1 may be combined with any second sequence shown in the same table. An exemplary fusion spacer is shown in Table 2. The target sequence that binds to SCN1A exon 20N may include, essentially, or may be essentially, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences shown in the table below.

[0085] (Table 2) Exon 20N targeting sequence (fusion spacer) TIFF2026536590000003.tif191161TIFF2026536590000004.tif110161

[0086] In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 52. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 53. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 54. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 55. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 56. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 57. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 58. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 59. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 60. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 61. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 62. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 63. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 64. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 65. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 66. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 67.In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 68. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 69. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 70. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 71. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 72. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 73. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 74. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 75. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 76. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 77. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 78. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 79. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 80. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 81. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 82. In some embodiments, the target sequence that binds to SCN1A exon 20N includes the sequence shown in SEQ ID NO: 83.

[0087] In some embodiments, the snRNAs of this disclosure target a premRNA or mRNA sequence encoding a protein that is synaptic Ras GTPase activating protein 1. SYNGAP1 is a gene that encodes a protein called synaptic Ras GTPase activating protein 1 (SYNGAP1). Mutations in SYNGAP1 are associated with SYNGAP1-associated epilepsy. In some embodiments, the SYNGAP1 RNA sequence targeted by the snRNA composition of this disclosure may be any SYNGAP1 exon or SYNGAP1 intron RNA sequence. In some embodiments, the SYNGAP1 RNA sequence targeted by the snRNA composition of this disclosure is an exon 11 or harmful exon 11 SYNGAP1 RNA sequence. In some embodiments, the SYNGAP1 RNA sequence targeted by the snRNA composition of this disclosure is a human exon 11 or harmful exon 11 SYNGAP1 RNA sequence. In some embodiments, the SYNGAP1 RNA sequence targeted by the snRNA composition of the Disclosure is a mouse exon 11 or harmful exon 11 SCN1A RNA sequence. In some embodiments, the SYNGAP1 RNA sequence targeted by the snRNA of the Disclosure is a splice acceptor sequence, a splice donor sequence, or an exon splice enhancer sequence.

[0088] In some embodiments, the nucleic acid sequence encoding human wild-type SYNGAP1 mRNA includes or consists of SEQ ID NO: 280. In some embodiments, the nucleic acid sequence encoding human wild-type SCN1A mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 280.

[0089] To prepare a fusion spacer, the sequences shown in Table 3 may be combined. Any first sequence shown in Table 3 may be combined with any second sequence shown in Table 3. Exemplary fusion spacers are shown in Table 3. The target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 may include, be essentially, or be derived from, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences shown in Table 3 below.

[0090] (Table 3) Exon 20N targeting sequences and exon 20N targeting fusion sequences TIFF2026536590000005.tif83161TIFF2026536590000006.tif236161TIFF20265365900 00007.tif236161TIFF2026536590000008.tif239161TIFF2026536590000009.tif50161

[0091] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 181. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 182. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 183. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 184. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 185. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 186. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 187. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 188. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 189. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 190. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 191. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 192. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 193.In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 194. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 195. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 196. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 197. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 198. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 199. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 200.

[0092] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 201. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 202. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 203. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 204. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 205. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 206. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 207. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 208. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 209. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 210.

[0093] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 211. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 212. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 213. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 214. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 215. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 216. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 217. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 218. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 219. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 220.

[0094] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 221. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 222. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 223. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 224. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 225. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 226. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 227. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 228. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 229. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 230.

[0095] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 231. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 232. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 233. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 234. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 235. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 236. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 237. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 238. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 239. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 240.

[0096] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 241. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 242. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 243. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 244. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 245. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 246. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 247. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 248. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 249. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 250.

[0097] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 251. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 252. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 253. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 254. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 255. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 256. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 257. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 258. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 259. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 260.

[0098] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 261. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 262. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 263. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 264. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 265. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 266. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 267. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 268. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 269. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 270.

[0099] In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 271. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 272. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 273. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 274. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 275. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 276. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 277. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 278. In some embodiments, the target sequence that binds to SYNGAP1 exon 11 or harmful exon 11 includes the sequence shown in SEQ ID NO: 279.

[0100] stem loop The engineered snRNA (esnRNA) systems disclosed herein may include a stem-loop (SL), which includes compensatory modifications to the native snRNA stem-loop. These modifications result in increased stability of the engineered nuclear small ribonucleoprotein complex (esnRNP) compared to snRNPs containing an unmodified stem-loop. The SL disclosed herein may be derived from any snRNP, for example, U1-U12. In one embodiment, the SL is a human or mouse U7 SL. In one embodiment, the SL is a human SL. In one embodiment, the SL is a mouse SL. In several embodiments, the SL is a human and mouse SL. In several embodiments, the SL is a non-human SL, which is, for example, a mouse SL, a pig SL, a sheep SL, a goat SL, a cattle SL, a dog SL, a cattle SL, a horse SL, or a combination thereof. In several embodiments, the SL sequence is not a native stem-loop sequence. In some embodiments, the nucleic acid sequence of SL is not a natural stem-loop sequence by at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between). The stem-loop sequence is described in WO2023168458, and with respect to examples of SL sequences that may be used in the constructs described herein, the contents of that document are incorporated herein by reference in their entirety.

[0101] In some embodiments, human SL contains, is essentially, or consists of nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000010.tif88128.

[0102] In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 84. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 85. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 86. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 87. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 88. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 89. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 90. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 91. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 92. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 93. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 94. In some embodiments, the human SL includes the sequence shown in SEQ ID NO: 95.

[0103] In some embodiments, mouse SL contains, is essentially, or consists of nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000011.tif50136.

[0104] In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 96. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 97. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 98. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 98. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 100. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 101. In some embodiments, mouse SL contains the sequence shown in SEQ ID NO: 102.

[0105] In some embodiments, human or mouse SL contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000012.tif20140.

[0106] In some embodiments, the human or mouse SL contains the sequence shown in SEQ IID NO: 103. In some embodiments, the human or mouse SL contains the sequence shown in SEQ IID NO: 104. In some embodiments, the human or mouse SL contains the sequence shown in SEQ IID NO: 105.

[0107] In some embodiments, canine or feline SL contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the following nucleotide sequences: TIFF2026536590000013.tif5128.

[0108] In some embodiments, cattle, sheep, or goat SL contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000014.tif12137

[0109] . In some embodiments, the cattle, sheep, or goat SL contains the sequence shown in SEQ ID NO: 107. In some embodiments, the cattle, sheep, or goat SL contains the sequence shown in SEQ ID NO: 108.

[0110] In some embodiments, porcine SL contains, is essentially, or consists of nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000015.tif35140.

[0111] In some embodiments, the pig SL contains the sequence shown in SEQ ID NO: 109. In some embodiments, the pig SL contains the sequence shown in SEQ ID NO: 110. In some embodiments, the pig SL contains the sequence shown in SEQ ID NO: 111. In some embodiments, the pig SL contains the sequence shown in SEQ ID NO: 112. In some embodiments, the pig SL contains the sequence shown in SEQ ID NO: 113.

[0112] In some embodiments, horse SL contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000016.tif12137.

[0113] In some embodiments, the horse SL contains the sequence shown in SEQ ID NO: 114. In some embodiments, the horse SL contains the sequence shown in SEQ ID NO: 115.

[0114] In some embodiments, sheep SL contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000017.tif20140.

[0115] In some embodiments, sheep SL contains the sequence shown in SEQ ID NO: 116. In some embodiments, sheep SL contains the sequence shown in SEQ ID NO: 117. In some embodiments, sheep SL contains the sequence shown in SEQ ID NO: 118.

[0116] In some embodiments, the stem-loop results in increased stability of the snRNA compared to snRNAs containing a native stem-loop. In some embodiments, the native snRNA stem-loop contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences: TIFF2026536590000018.tif42137.

[0117] In some embodiments, the natural snRNA stem-loop contains the sequence shown in SEQ ID NO: 119. In some embodiments, the natural snRNA stem-loop contains the sequence shown in SEQ ID NO: 120. In some embodiments, the natural snRNA stem-loop contains the sequence shown in SEQ ID NO: 121. In some embodiments, the natural snRNA stem-loop contains the sequence shown in SEQ ID NO: 122.

[0118] 5' interaction stabilization domain The SLs disclosed herein have more effective folding and annealing properties due to the 5' interaction stabilization domain (5'ISD), which in turn results in increased esnRNA stability compared to unmanipulated snRNA. The 5'ISD has nucleotides complementary to the nucleotides in the SL, and, without wishing to be bound by theory, the interaction between the 5'ISD and the SL is predicted to form a secondary structure that protects the 5' end of the snRNA. In some aspects, the 5'ISD anneals and / or hybridizes to the SLs of this disclosure. In some aspects, the 5'ISD is a sequence that is complementary and / or reverse complementary to the sequence present in the SLs of this disclosure. In some aspects, the 5'ISDs disclosed herein may include or consist of one of the following nucleotide sequences: TIFF2026536590000019.tif88128.

[0119] Sm-joint domain The snRNA systems disclosed herein utilize an Sm-binding domain (SmBD). The ring of Sm proteins that assemble around the Sm-binding domain (SmBD) to form snRNPs includes SmB / B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. The Sm-binding site of U7 recruits an endogenous RNA-binding factor and can be replaced with a non-U7 snRNA to produce a more stable esnRNA. In one embodiment, the SmBD is U1 SmBD, U2 SmBD, U4 SmBD, or SmBD. In another embodiment, the SmBD is derived from a pseudosnRNA. In another embodiment, the SmBD is a nucleotide sequence containing ATTTTT. In another embodiment, the SmBD includes the following nucleotide sequence: TIFF2026536590000020.tif19156.

[0120] Promoter array The gene therapy compositions and RNA-targeting snRNA gene therapy compositions of this disclosure include promoter sequences derived from snRNA. A “promoter” is a regulatory sequence, which is a region of polynucleotide sequence where the initiation and rate of transcription are controlled. It may include a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind.

[0121] The snRNA systems disclosed herein may include an snRNA promoter derived from any of U1 to U12. In one embodiment, the snRNA promoter is the U7 promoter. In another embodiment, the U7 promoter is the human U7 promoter (hU7) or the mouse U7 promoter (mU7). In yet another embodiment, the U7 promoter is the human endogenous U7 promoter, which is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the following SEQ ID NO: 135: TIFF2026536590000021.tif40158.

[0122] In one embodiment, the snRNA promoter is the U1 promoter. In another embodiment, the U1 promoter is the human U1 promoter or the mouse U1 promoter.

[0123] In another embodiment, the same snRNA promoter drives the expression of each copy of the snRNA insert. In another embodiment, each copy of the snRNA insert is identical. In another embodiment, different snRNA promoters drive each copy of the snRNA insert. In one embodiment, 2x snRNA includes a mouse U7 promoter that drives one copy of the snRNA insert and a mouse U1 promoter that drives the other copy of the snRNA insert.

[0124] In other contexts, the snRNA promoter is either the PolII promoter or the PolIII promoter.

[0125] In other contexts, the snRNA promoter contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the promoters and / or promoter sequences listed in Table 4 below.

[0126] (Table 4) Exemplary promoter sequences TIFF2026536590000022.tif104161TIFF2026536590000023.tif232161TIFF2026536590000024.tif237161TIFF2026536590000025.tif99161

[0127] In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 125. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 126. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 127. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 128. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 129. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 130. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 131. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 132. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 133. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 134. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 135. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 136. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 137. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 138. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 139. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 281. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 282. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 283. In some embodiments, the snRNA promoter includes the sequence shown in SEQ ID NO: 284.

[0128] Terminator array The snRNA systems disclosed herein may include a downstream terminator (DT) of the snRNA. The downstream terminator defines the end of a transcription unit, such as an esnRNA or snRNA. In another embodiment, the snRNA DT is a U7 DT comprising the following sequence: TIFF2026536590000026.tif12159.

[0129] In one embodiment, the snRNA comprises an SL or eSL, one or more promoters, a TS targeting an SCN1A or SYNGAP1 RNA molecule, a SmBD, a 5'ISD, and a DT. In another embodiment, the snRNA comprises a native stem-loop, one or more promoters, a TS targeting an SCN1A or SYNGAP1, a SmBD, and a DT. The sequences of promoters and DTs provided herein can be successfully combined in any combination.

[0130] In some embodiments, DT comprises, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the DT sequences listed in Table 5 below.

[0131] (Table 5) Exemplary DT sequences TIFF2026536590000027.tif85161TIFF2026536590000028.tif218161

[0132] In some embodiments, DT includes the sequence shown in SEQ ID NO: 140. In some embodiments, DT includes the sequence shown in SEQ ID NO: 141. In some embodiments, DT includes the sequence shown in SEQ ID NO: 142. In some embodiments, DT includes the sequence shown in SEQ ID NO: 143. In some embodiments, DT includes the sequence shown in SEQ ID NO: 144. In some embodiments, DT includes the sequence shown in SEQ ID NO: 145. In some embodiments, DT includes the sequence shown in SEQ ID NO: 146. In some embodiments, DT includes the sequence shown in SEQ ID NO: 147. In some embodiments, DT includes the sequence shown in SEQ ID NO: 148. In some embodiments, DT includes the sequence shown in SEQ ID NO: 149. In some embodiments, DT includes the sequence shown in SEQ ID NO: 150. In some embodiments, DT includes the sequence shown in SEQ ID NO: 285. In some embodiments, DT includes the sequence shown in SEQ ID NO: 286. In some embodiments, DT includes the sequence shown in SEQ ID NO: 287. In some embodiments, DT includes the sequence shown in SEQ ID NO: 288. In some embodiments, DT includes the sequence shown in SEQ ID NO: TTTTTT.

[0133] In one embodiment, snRNA is delivered via an AAV vector.

[0134] In some embodiments, the AAV vector contains multiple copies of snRNA. In some embodiments, the multiple copies of snRNA are 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of (2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x) snRNA. In some embodiments, the multiple copies of snRNA are 4 or more copies of snRNA.

[0135] In some embodiments, each snRNA, which is a multiple copy of an snRNA, is separated by a nucleic acid buffer sequence derived from a human non-coding genome sequence downstream of the snRNA. In one embodiment, the buffer sequence is derived from a human genome sequence downstream of U7.

[0136] In one embodiment, the buffer sequence is one of the following nucleic acid sequences: TIFF2026536590000029.tif49144

[0137] In another embodiment, the buffer sequence is one of the following nucleic acid sequences: TIFF2026536590000030.tif63152

[0138] In another embodiment, the buffer sequence is one of the following nucleic acid sequences: TIFF2026536590000031.tif180153

[0139] The 100 bp and 500 bp buffer 1 sequences originate from a sequence beginning 100 bp downstream of the mouse (mus musculus) U7 pseudogene 8 (location chromosome 14: 4,409,359~4,409,421 reverse strand, GRCm39:CM001007.3). The 100 bp and 500 bp buffer 2 sequences originate from a sequence beginning 130 bp downstream of the human U7 pseudogene 5 (X chromosome: 140,451,148~140,451,208 forward strand, GRCh38:CM000685.2). Both 100 bp buffer sequences are the first 100 bp of their corresponding 500 bp buffer sequences (for example, "buffer 1 (100 bp)" consists of the first 100 bp of "buffer 1 (500 bp)"). The 30 bp buffers 1, 2, and 3 are consecutive 30 bp sequences within the "100 bp buffer 1" downstream of the mouse U7 pseudogene 8. These downstream sequences were selected based on the following criteria: they do not contain any known regulatory sites or regulatory genes (using Gencode / Ensembl) within or near them; they do not contain repetitive sequences; they have a 40-60% GC content for the entire buffer; they have a 40-60% GC content in the 20 bp regions at both ends of the buffer; and they have minimal sequence complexity.

[0140] snRNA sequence The snRNA sequences disclosed herein may include any combination of the characteristics of esnRNA or snRNA described herein.

[0141] In some embodiments, the snRNA comprises a targeting sequence that binds to the SCN1A RNA sequence and a SL. In some embodiments, the SCN1A targeting sequence comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 16-83. In some embodiments, the SL comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 84-124. In some embodiments, the SL comprises or consists of the nucleic acid sequence shown in SEQ ID NO: 97.

[0142] In some embodiments, the snRNA includes a targeting sequence that binds to an SCN1A RNA sequence, comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 16-83, and an SL comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 84-124. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes a nucleic acid sequence shown in any one of SEQ ID NO: 16-83, and the SL includes a nucleic acid sequence shown in SEQ ID NO: 97.

[0143] In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 16, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 17, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 18, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 19, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 20, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 21, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 22, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 23, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 24, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 25, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 26, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 27, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 28, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 29, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 30, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 31, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 32, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 33, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 34, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 35, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 36, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 37, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 38, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 39, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 40, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 41, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 42, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 43, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 44, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 45, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 46, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 47, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 48, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 49, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 50, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 51, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 52, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 53, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 54, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 55, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 56, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 57, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 58, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 59, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 60, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 61, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 62, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 63, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 64, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 65, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 66, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 67, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 68, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 69, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 70, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 71, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 72, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 73, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 74, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 75, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 76, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 77, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 78, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 79, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 80, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 81, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, including the nucleic acid sequence shown in SEQ ID NO: 82, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the snRNA includes a targeting sequence that binds to the SCN1A RNA sequence, which includes the nucleic acid sequence shown in SEQ ID NO: 83, and the SL includes the nucleic acid sequence shown in SEQ ID NO: 97.

[0144] In some embodiments, the SCN1A targeting sequence is located 5' to the SL.

[0145] Polynucleotides and vectors This specification also provides polynucleotides and vectors (e.g., recombinant expression vectors) containing snRNAs targeting SCN1A or SYNGAP1.

[0146] In some embodiments of the compositions and methods of this disclosure, polynucleotides or vectors comprising an snRNA system targeting SCN1A or SYNGAP1 are provided herein. In some embodiments of the compositions and methods of this disclosure, polynucleotides or vectors comprising an snRNA system targeting SCN1A are provided herein. In some embodiments of the compositions and methods of this disclosure, polynucleotides or vectors comprising an snRNA system targeting SYNGAP1 are provided herein. In some embodiments, the vector is a single vector or a unit vector.

[0147] This specification provides polynucleotides or vectors comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to a nucleic acid sequence shown in any one of SEQ ID NO: 162 to 180. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 162. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 163. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 164. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 165. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 166. In some embodiments, the polynucleotide or vector comprises the nucleic acid sequence shown in SEQ ID NO: 167. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 168. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 169. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 170. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 171. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 172. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 173. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 174. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 175. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 176.In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 177. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 178. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 179. In some embodiments, the polynucleotide or vector includes the nucleic acid sequence shown in SEQ ID NO: 180.

[0148] In some embodiments, the snRNA system can target one or more SCN1A RNA sequences. In some aspects, the SCN1A RNA sequence is an SCN1A premRNA sequence. In some embodiments, the snRNA system can target one or more SYNGAP1 RNA sequences. In some aspects, the SYNGAP1 RNA sequence is a SYNGAP1 premRNA sequence. In some aspects, the snRNA system can target multiple (i.e., two or more) RNAs of interest. In some embodiments, the two or more RNAs of interest may be sequences that are identical premRNA molecules but differ within the premRNA molecule.

[0149] One type of vector is a "plasmid," which refers to a circular, double-stranded DNA loop into which, for example, standard molecular cloning techniques can insert DNA segments in addition to the nucleotide of interest. Another type of vector is a viral vector, which contains a viral DNA or RNA sequence for packaging into a virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses). Viral vectors also contain polynucleotides carried by the virus for transfection into host cells. In some embodiments, the vector is a lentiviral vector (e.g., an integration-deficient lentiviral vector) or an adeno-associated virus (AAV) vector. Vectors, such as bacterial vectors with bacterial origins of replication and episomal mammalian vectors, may be capable of autonomous replication in the host cell into which they are introduced, while other vectors, such as non-episomal mammalian vectors, are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome.

[0150] In some embodiments, vectors, such as expression vectors, can direct the expression of the genes they contain. Common expression vectors often take the form of plasmids. In some embodiments, recombinant expression vectors include nucleic acids provided herein, such as esnRNA in a form suitable for the expression of RNA molecules in host cells. Recombinant expression vectors may include one or more regulatory elements that can be selected based on the host cell to be used for expression, the regulatory elements being functionally ligated to the nucleic acid sequence to be expressed. "Functionally ligated" in a recombinant expression vector is intended to mean that the nucleotide sequence of interest is ligated to the regulatory element in a manner that enables the expression of the nucleotide sequence, for example, in an in vitro transcription / translation system or in a host cell when the vector has been introduced into the host cell. In some embodiments, the regulatory element is a promoter as described herein. In some embodiments, the regulatory element is a terminator as provided herein.

[0151] Specific embodiments of the vector vary depending on factors such as the selection of host cells to be transformed and the desired expression level. The vector may be introduced into host cells, thereby producing transcripts, proteins, or peptides, including fusion proteins or fusion peptides, encoded by the nucleic acids described herein, such as snRNA, CRISPR transcripts, proteins, enzymes, their variants, and their fusion proteins.

[0152] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, expression regulatory elements. “Expression regulatory element” means, as used herein, any sequence that regulates expression, such as a coding sequence, for example, a gene. Exemplary expression regulatory elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression regulatory elements may be, for example, constitutive, inductive, repressive, or tissue-specific. “Promoter” is one of the regulatory sequences, which is a polynucleotide sequence region where the initiation and rate of transcription are regulated. Promoters may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. “Enhancer” is a region of DNA to which an activated protein can bind, thereby increasing the likelihood or frequency of transcription.

[0153] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, vector elements such as buffer sequences derived downstream of the human genome sequence from the snRNA, which thus have the ability to encode multiple snRNAs derived from a single construct.

[0154] In some embodiments, the snRNA constructs disclosed herein include a bidirectional snRNA promoter for expressing the snRNA.

[0155] In another embodiment, the vector configuration may include a linker, a signal sequence, and / or a tag.

[0156] Viral vector In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector. In some embodiments, the vector is a retrovirus vector, an adenovirus / retrovirus chimeric vector, a herpes simplex virus type I or type II vector, a parvovirus vector, a reticuloendotheliosis virus vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector, or any hybrid or chimeric vector incorporating preferred aspects of two or more viral vectors.

[0157] In some embodiments, the vector further comprises one or more expression regulatory elements functionally linked to a polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector is not integrated into the host genome, thereby reducing the probability of causing insertional mutations. In some embodiments, the AAV vector may encode polynucleotides in the range of 4.5 kb to 4.75 kb in length. In some embodiments, exemplary AAV vectors that may be used in any of the compositions, systems, methods, and kits described herein include the AAV1 vector, modified AAV1 vector, AAV2 vector, modified AAV2 vector, AAV2-Tyr variant vector, AAV3 vector, modified AAV3 vector, AAV4 vector, modified AAV4 vector, AAV5 vector, modified AAV5 vector, AAV6 vector, modified AAV6 vector, AAV7 vector, modified AAV7 vector, AAV8 vector, AAV This may include rh8 vectors, AAV9 vectors, AAV.rh10 vectors, modified AAV.rh10 vectors, AAVrh.74 vectors, AAV.rh32 / 33 vectors, modified AAV.rh32 / 33 vectors, AAV.rh43 vectors, modified AAV.rh43 vectors, AAV.rh64R1 vectors, and modified AAV.rh64R1 vectors, AAV-Tyr mutant vectors, AAV-Tyr-Ser mutant vectors, AAV-Tyr-Ser-Thr mutant vectors, and any combination thereof or any equivalent thereof. In some embodiments, the lentiviral vectors are integrase-competent lentiviral vectors (ICLVs).In some embodiments, a lentiviral vector may refer to a transgene plasmid vector, as well as a transgene plasmid vector combined with an associated plasmid (e.g., a packaging plasmid, a rev expression plasmid, or an envelope plasmid), and may also refer to a lentivirus-based particle capable of introducing foreign nucleic acids into a cell via a viral or virus-like entry mechanism.

[0158] In some embodiments, the viral vector includes a sequence isolated from or derived from a retrovirus. In some embodiments, the viral vector includes a sequence isolated from or derived from a lentivirus. In some embodiments, the viral vector includes a sequence isolated from or derived from an adenovirus. In some embodiments, the viral vector includes a sequence isolated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is non-replicable. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0159] In some embodiments, the vector further comprises one or more expression regulatory elements functionally linked to a polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the vector has low toxicity. In some embodiments, the vector is not integrated into the host genome, thereby reducing the probability of causing insertional mutations.

[0160] lentiviral vectors Lentiviral vectors are well known in the art (see, for example, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg and Durand et al. (2011) Viruses 3(2):132-159 doi: 10.3390 / v3020132). In some embodiments, exemplary lentiviral vectors that may be used in any of the compositions, systems, methods, and kits described herein include human immunodeficiency virus (HIV) 1 vector, modified human immunodeficiency virus (HIV) 1 vector, human immunodeficiency virus (HIV) 2 vector, modified human immunodeficiency virus (HIV) 2 vector, and suutee mangabey-derived simian immunodeficiency virus (SIV). SM ) vector, modified Sutie Mangabei-derived simian immunodeficiency virus (SIV) SM ) Vector, simian immunodeficiency virus (SIV) derived from African green monkeys AGM ) vector, modified African green monkey-derived simian immunodeficiency virus (SIV) AGM This may include vectors such as equine infectious anemia virus (EIAV), modified equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), modified feline immunodeficiency virus (FIV), Visna maedivirus (VNV / VMV), modified Visna maedivirus (VNV / VMV), canine arthritis encephalitis virus (CAEV), modified canine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), or modified bovine immunodeficiency virus (BIV).

[0161] Adeno-associated virus vector In some respects, the vectors described herein are AAV viral vectors. The terms “adeno-associated virus” or “AAV,” as used herein, refer to a class of viruses associated with this name, and to a member of the class of viruses belonging to the Parvoviridae family, genus Dependoparvovirus. Adeno-associated viruses are single-stranded DNA viruses that replicate in cells where certain functions are provided by co-infecting helper viruses. General information and reviews on AAV can be found, for example, in: Carter, 1989, “Handbook of Parvoviruses”, Vol. 1, pp. 169–228, and Berns, 1990, “Virology”, pp. 1743–1764, Raven Press, (New York). It is quite foreseeable that the same principles described in these reviews are also applicable to further AAV serotypes that have been characterized since the publication date of those reviews, as it is well known that various serotypes are very closely related in both structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, "Parvoviruses and Human Disease", JR Pattison, ed., pp. 165-174; and Rose, "Comprehensive Virology" 3: 1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication characteristics, mediated by homologous rep genes; and they all carry three related capsid proteins, such as those expressed in AAV2. The degree of association has been further suggested by heteroduplex analysis, which has revealed extensive cross-hybridization between serotypes across the entire length of the genome, as well as the presence of similar self-annealing segments at the ends corresponding to “inverted terminal repeats” (ITRs).The similar infectivity patterns also suggest that the replication function in each serotype is under similar regulatory control. Several serotypes of this virus are known to be suitable for gene delivery; all known serotypes are capable of infecting cells from various tissue types.

[0162] AAV possesses unique characteristics that make it of interest as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection in cultured cells is non-cytotoxic, and spontaneous infection in humans and other animals is asymptomatic. Furthermore, because AAV infects many mammalian cells, it has the potential to target many different tissues in vivo. Additionally, AAV can be transmitted to slow-dividing and non-dividing cells and, essentially, can persist for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in a plasmid, enabling the construction of a recombinant genome. Furthermore, since the signals directing AAV replication and capsid inclusion of the genome are contained within the ITR of the AAV genome, it is possible to construct an AAV vector by replacing some or all of approximately 4.3 kb of the genome (encoding the replication protein and capsid structure protein, rep-cap) with foreign DNA. The rep and cap proteins may be provided in trans. Another notable characteristic of AAV is that it is an extremely stable and robust virus. Since AAV readily withstands the conditions used to inactivate adenoviruses (56°C–65°C for several hours), storing AAV at low temperatures is not particularly important. AAV can even be freeze-dried. Finally, cells infected with AAV are not resistant to reinfection.

[0163] The recombinant AAV (rAAV) genome of the present invention may comprise, essentially comprise, or be derived from, a nucleic acid molecule encoding at least one esnRNA and one or more AAV ITRs adjacent to said nucleic acid molecule. The construction of pseudotyped rAAVs is disclosed, for example, in WO2001083692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). Nucleotide sequences of genomes of various AAV serotypes are known in the art.

[0164] The AAV vectors described herein may comprise, essentially consist of, or consist of, one or more nucleic acid molecules and one or more AAV ITRs. In some aspects, the nucleic acid molecules encode the esnRNA of this disclosure. Such AAV vectors can be replicated and packaged into infectious viral particles, for example, when present in a host cell providing functionality for rep and cap gene products by transfection into the host cell. In some aspects, the AAV vector comprises a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or an enhancer and / or terminator, flanked by ITRs on both sides, and packaged within an infectious AAV particle. The nucleic acid portion encapsulated within the capsid may be referred to as the AAV vector genome. Plasmids containing the AAV vector may also include elements for manufacturing purposes, such as antibiotic resistance genes, origin sequences, etc., but these are not encapsulated within the capsid and therefore do not form part of the AAV particle.

[0165] In some aspects, an AAV vector may include at least one nucleic acid encoding the esnRNA of the Disclosure. In some aspects, an AAV vector may include at least one regulatory sequence. In some aspects, an AAV vector may include at least one inverted end (ITR) sequence of AAV. In some aspects, an AAV vector may include a first ITR sequence and a second ITR sequence. In some aspects, an AAV vector may include at least one promoter sequence. In some aspects, an AAV vector may include at least one enhancer sequence. In some aspects, an AAV vector may include at least one terminator sequence. In some aspects, an AAV vector may include at least one poly(A) sequence. In some aspects, an AAV vector may include at least one linker sequence. In some aspects, an AAV vector may include at least one buffer sequence. In some aspects, an AAV vector of the Disclosure may include at least one nuclear localization signal or at least one nuclear export signal, and / or both. In some cases, the AAV vector may include a first AAV ITR sequence, a promoter sequence, an snRNA sequence and / or an esnRNA sequence, a terminator sequence, and a second AAV ITR sequence. In some cases, the AAV vector may include a first AAV ITR sequence, a promoter sequence, an esnRNA sequence, a terminator sequence, and a second AAV ITR sequence in the 5' to 3' direction. In some cases, the AAV vector may include a first AAV ITR sequence, a promoter sequence, an snRNA sequence, a terminator sequence, and a second AAV ITR sequence in the 5' to 3' direction.

[0166] In some cases, an AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, a stop sequence, a second promoter sequence, a second snRNA sequence, a second stop sequence, and a second AAV ITR sequence. In some cases, an AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, a stop sequence, a second promoter sequence, a second snRNA sequence, a second stop sequence, a third promoter sequence, a third snRNA sequence, a third stop sequence, and a second AAV ITR sequence. In some aspects, the AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, a stop sequence, a second promoter sequence, a second snRNA sequence, a second stop sequence, a third promoter sequence, a third snRNA sequence, a third stop sequence, a fourth promoter sequence, a fourth snRNA sequence, a fourth stop sequence, and a second AAV ITR sequence.

[0167] In some aspects of the compositions and methods of this disclosure, the viral vector comprises a sequence isolated from or derived from adeno-associated virus (AAV). In some aspects, the viral vector comprises an ITR sequence or a capsid sequence isolated from or derived from AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12. In some aspects, the AAV serotype is AAVrh.74. In one aspect, the AAV vector comprises a modified capsid. In one aspect, the AAV vector is an AAV2-Tyr variant vector. In one embodiment, the AAV vector comprises a capsid having a non-tyrosine amino acid at a position corresponding to a surface-exposed tyrosine residue, such as the positions of Tyr252, Tyr272, Tyr275, Tyr281, Tyr508, Tyr612, Tyr704, Tyr720, Tyr730, or Tyr673 in wild-type AAV2. See also WO 2008 / 124724, the entirety of which is incorporated herein by reference. In some embodiments, the AAV vector comprises an engineered capsid. AAV vectors comprising an engineered capsid include, but are not limited to, AAV2.7m8, AAV9.7m8, AAV2 2tYF, and AAV8 Y733F. In some embodiments, the capsid is a ubiquitination-resistant capsid. In another embodiment, the ubiquitinated capsid is an AAV2 capsid containing tyrosine (Y) and serine (S) mutations. In another embodiment, the AAV2 capsid contains Y, S, and threonine (T) mutations. In another embodiment, the AAV2 capsid includes, but is not limited to, AAV2 capsid variants such as T455V, T491V, T550V, T659V, Y444+500+730F, and Y444+500+730F+T491V. In some embodiments, the viral vector is non-replicable. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).In some embodiments, the viral vector is single-stranded (ssAAV).

[0168] In some embodiments, the snRNAs provided herein are contained within single-stranded AAV (ssAAV). In some embodiments, the snRNAs provided herein are contained within self-complementary AAV (scAAV). Due to the single-stranded nature of the parvovirus genome, cellular mechanisms are required to provide a complementary strand for gene expression. This recruitment activity in cells is considered the rate-limiting factor in the efficiency of transduction and gene expression in parvoviruses and parvovirus particles. By using scAAV instead of ssAAV, this well-known problem is resolved by packaging both strands as a single double-stranded DNA molecule (or inverted repeat genome) that can be folded into dsDNA due to the self-complementary viral genome sequence. In this respect, the need for DNA synthesis, or the need for base pairing between multiple viral genomes, is eliminated.

[0169] AAV ITR array In some embodiments of the compositions and methods of the present disclosure, the AAV inverted terminal repeat sequence may include any AAV ITR sequence known in the art. In some aspects, the AAV ITR sequence includes or consists of: AAV1 ITR sequence, AAV2 ITR sequence, AAV3 ITR sequence, AAV4 ITR sequence, AAV5 ITR sequence, AAV6 ITR sequence, AAV7 ITR sequence, AAV8 ITR sequence, AAV9 ITR sequence, AAV10 ITR sequence, AAVrh10 ITR sequence, AAV11 ITR sequence, AAV12 ITR sequence, AAV13 ITR sequence, or AAVrh74 ITR sequence.

[0170] In some aspects, the ITR sequence may include a modified AAV ITR sequence.

[0171] In some aspects, the AAV ITR sequence may contain, be essentially, or be essentially, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 158 or SEQ ID NO: 159.

[0172] In some embodiments, the AAV vectors provided herein include first and second AAV ITR sequences. In some aspects, the first AAV ITR sequence may include, essentially, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to SEQ ID NO: 158 or SEQ ID NO: 159, and the second AAV ITR sequence may include, essentially, or may consist of, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to SEQ ID NO: 158 or SEQ ID NO: 159. In some cases, the first AAV ITR sequence is located on the 5' side of the AAV vector. In some cases, the second AAV ITR sequence is located on the 3' side of the AAV vector.

[0173] In some aspects, the first AAV ITR sequence includes the sequence indicated by SEQ ID NO: 158 or SEQ ID NO: 159. In some aspects, the second AAV ITR sequence includes the sequence indicated by SEQ ID NO: 158 or SEQ ID NO: 159. In some embodiments, the AAV vector provided herein includes a first AAV ITR sequence containing the sequence indicated by SEQ ID NO: 158, and a second AAV ITR sequence containing the sequence indicated by SEQ ID NO: 59. In some aspects, the first AAV ITR sequence is located on the 5' side of the AAV vector. In some aspects, the second AAV ITR sequence is located on the 3' side of the AAV vector.

[0174] In some embodiments of the compositions and methods of this disclosure, the viral vector comprises a sequence isolated from or derived from adeno-associated virus (AAV).

[0175] In some embodiments of the compositions and methods of this disclosure, the vectors of this disclosure are nonviral vectors. In some embodiments, the vectors comprise or consist of nanoparticles, micelles, liposomes or lipoplexes, polymerosomes, polyplexes, or dendrimers. In some embodiments, the vectors are expression vectors or recombinant expression systems. As used herein, the term “recombinant expression system” refers to a genetic construct for expressing any genetic material formed by recombinantity.

[0176] snRNA vector construct This specification also provides vector constructs targeting SCN1A, including the snRNA constructs described herein. This specification also provides vector constructs targeting SYNGAP1, including the snRNA constructs described herein.

[0177] An exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05290. The elements of A05290 are shown in Table 6. In some aspects, the nucleic acid sequence encoding the AAV vector A05290 includes SEQ ID NO: 162.

[0178] (Table 6) A05290:scAAV-2x_mU7prom-SCN1A1z15 / z6 -mU7term_mU1prom- SCN1A1z15 / z6 -mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000032.tif229166

[0179] The nucleotide sequence of A05290 (the entire transgene from ITR to ITR): TIFF2026536590000033.tif165160

[0180] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05291. The elements of A05291 are shown in Table 7. In some aspects, the nucleic acid sequence encoding the AAV vector A05291 includes SEQ ID NO: 163.

[0181] (Table 7) A05291:scAAV-2x_mU7prom-SCN1A1z11 / z15-mU7term_mU1prom- SCN1A1z11 / z15-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000034.tif221166

[0182] Nucleotide sequence of A05291 (entire transgene from ITR to ITR): TIFF2026536590000035.tif165160

[0183] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05378. The elements of A05378 are shown in Table 8. In some aspects, the nucleic acid sequence encoding the AAV vector A05378 includes SEQ ID NO: 164.

[0184] (Table 8) A05378:scAAV-2x_mU7prom-SCN1A1z12 / z6 -mU7term_mU1prom- SCN1A1z12 / z6 -mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000036.tif229166

[0185] The nucleotide sequence of A05378 (the entire transgene from ITR to ITR): TIFF2026536590000037.tif165160

[0186] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05379. The elements of A05379 are shown in Table 9. In some aspects, the nucleic acid sequence encoding the AAV vector A05379 includes SEQ ID NO: 165.

[0187] (Table 9) A05379: scAAV-2x_mU7prom-SCN1A1z33 / z15 -mU7term_mU1prom- SCN1A1z33 / z15 -mU1term; mouse loop with 5'ISD and eSL; nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000038.tif229166

[0188] The nucleotide sequence of A05379 (the entire transgene from ITR to ITR): TIFF2026536590000039.tif164160

[0189] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05380. The elements of A05380 are shown in Table 10. In some aspects, the nucleic acid sequence encoding the AAV vector A05380 includes SEQ ID NO: 166.

[0190] (Table 10) A05380:scAAV-2x_mU7prom-SCN1A1z15 / z33-mU7term_mU1prom-SCN1A1z15 / z33-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000040.tif229166

[0191] The nucleotide sequence of A05380 (the entire transgene from ITR to ITR): TIFF2026536590000041.tif165159

[0192] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05381. The elements of A05381 are shown in Table 11. In some aspects, the nucleic acid sequence encoding the AAV vector A05381 includes SEQ ID NO: 167.

[0193] (Table 11) A05381:scAAV-2x_mU7prom-SCN1A1z33 / z36-mU7term_mU1prom-SCN1A1z33 / z36-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000042.tif229166

[0194] The nucleotide sequence of A05381 (the entire transgene from ITR to ITR): TIFF2026536590000043.tif165159

[0195] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05382. The elements of A05382 are shown in Table 12. In some aspects, the nucleic acid sequence encoding the AAV vector A05382 includes SEQ ID NO: 168.

[0196] (Table 12) A05382:scAAV-2x_mU7prom-SCN1A1z36 / z33-mU7term_mU1prom- SCN1A1z36 / z33-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000044.tif229166

[0197] The nucleotide sequence of A05382 (the entire transgene from ITR to ITR): TIFF2026536590000045.tif165160

[0198] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05383. The elements of A05383 are shown in Table 13. In some aspects, the nucleic acid sequence encoding the AAV vector A05383 includes SEQ ID NO: 169.

[0199] (Table 13) A05383:scAAV-2x_mU7prom-SCN1A1z21 / z33-mU7term_mU1prom-SCN1A1z21 / z33-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000046.tif229166

[0200] The nucleotide sequence of A05383 (the entire transgene from ITR to ITR): TIFF2026536590000047.tif165159

[0201] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05384. The elements of A05384 are shown in Table 14. In some aspects, the nucleic acid sequence encoding the AAV vector A05384 includes SEQ ID NO: 170.

[0202] (Table 14) A05384:scAAV-2x_mU7prom-SCN1A1z33 / z21-mU7term_mU1prom-SCN1A1z33 / z21-mU1term; Mouse loop with 5'ISD and eSL; Nucleotide sequences of plasmid elements in order from N-terminus to C-terminus. TIFF2026536590000048.tif229166

[0203] The nucleotide sequence of A05384 (the entire transgene from ITR to ITR): TIFF2026536590000049.tif165159

[0204] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05865. The elements of A05865 are shown in Table 15. In some aspects, the nucleic acid sequence encoding the AAV vector A05865 includes SEQ ID NO: 171.

[0205] (Table 15) A05865:scAAV-2x_mU7prom-SCN1A1z15 / z6-mU7term_mU1prom-SCN1A1z15 / z6-mU1term; (Mouse loop without 5'ISD but with eSL) (Equivalent to synthetic SR220517); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000050.tif218166

[0206] The nucleotide sequence of A05865 (the entire transgene from ITR to ITR): TIFF2026536590000051.tif164159

[0207] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05866. The elements of A05866 are shown in Table 16. In some aspects, the nucleic acid sequence encoding the AAV vector A05866 includes SEQ ID NO: 172.

[0208] (Table 16) A05866:scAAV-2x_mU7prom-SCN1A1z11 / z15 -mU7term_mU1prom- SCN1A1z11 / z15 -mU1term; (Mouse loop with eSL but lacking 5'ISD) (Equivalent to synthetic SR220518); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus; Nucleotide sequence of plasmid element in order from N-terminus to C-terminus TIFF2026536590000052.tif210166

[0209] The nucleotide sequence of A05866 (the entire transgene from ITR to ITR): TIFF2026536590000053.tif157160

[0210] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05867. The elements of A05867 are shown in Table 17. In some aspects, the nucleic acid sequence encoding the AAV vector A05867 includes SEQ ID NO: 173.

[0211] (Table 17) A05867:scAAV-2x_mU7prom-SCN1A1z15 / z6 mU7term_mU1prom- SCN1A1z15 / z6 -mU1term; (Mouse loop without 5'ISD and eSL) (Equivalent to synthetic SR220520); Nucleotide sequence of plasmid element from N-terminus to C-terminus. TIFF2026536590000054.tif218166

[0212] The nucleotide sequence of A05867 (the entire transgene from ITR to ITR): TIFF2026536590000055.tif165160

[0213] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05868. The elements of A05868 are shown in Table 18. In some aspects, the nucleic acid sequence encoding the AAV vector A05868 includes SEQ ID NO: 174.

[0214] (Table 18) A05868:scAAV-2x_mU7prom-SCN1A1z11 / z15 -mU7term_mU1prom- SCN1A1z11 / z15 -mU1term; (Mouse loop without 5'ISD and eSL) (Equivalent to synthetic SR220521); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000056.tif210166

[0215] The nucleotide sequence of A05868 (the entire transgene from ITR to ITR): TIFF2026536590000057.tif157160

[0216] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A06047. The elements of A06047 are shown in Table 19. In some aspects, the nucleic acid sequence encoding the AAV vector A06047 includes SEQ ID NO: 175.

[0217] (Table 19) A06047: scAAV-2x_mU7prom-SCN1A1z15 / z6 -mU7term_mU1prom- SCN1A1z15 / z6 -mU1term; (Mouse loop without 5'ISD and eSL, short SL) (Equivalent to synthetic SR220590); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000058.tif218166

[0218] The nucleotide sequence of A06047 (the entire transgene from ITR to ITR): TIFF2026536590000059.tif165160

[0219] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A06048. The elements of A06048 are shown in Table 20. In some aspects, the nucleic acid sequence encoding the AAV vector A06048 includes SEQ ID NO: 176.

[0220] (Table 20) A06048: scAAV-2x_mU7prom-SCN1A1z11 / z15 -mU7term_mU1prom- SCN1A1z11 / z15 -mU1term; (Mouse loop without 5'ISD and eSL, short SL) (Equivalent to synthetic SR220591); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000060.tif210166

[0221] The nucleotide sequence of A06048 (the entire transgene from ITR to ITR): TIFF2026536590000061.tif157160

[0222] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05869. The elements of A05869 are shown in Table 21. In some aspects, the nucleic acid sequence encoding the AAV vector A05869 includes SEQ ID NO: 177.

[0223] (Table 21) A05869:scAAV-2x_mU7prom-SCN1A1z15 / z6 -mU7term_mU1prom- SCN1A1z15 / z6-mU1term; (Sheep loop without 5'ISD and eSL, long) (Equivalent to synthetic SR220523); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000062.tif227166

[0224] The nucleotide sequence of A05869 (the entire transgene from ITR to ITR): TIFF2026536590000063.tif165160

[0225] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A05870. The elements of A05870 are shown in Table 22. In some aspects, the nucleic acid sequence encoding the AAV vector A05870 includes SEQ ID NO: 178.

[0226] (Table 22) A05870:scAAV-2x_mU7prom-SCN1A1z11 / z15 -mU7term_mU1prom- SCN1A1z11 / z15 -mU1term; (Sheep loop without 5'ISD and eSL, long) (Equivalent to synthetic SR220524); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000064.tif219166

[0227] The nucleotide sequence of A05870 (the entire transgene from ITR to ITR): TIFF2026536590000065.tif166160

[0228] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A06049. The elements of A06049 are shown in Table 23. In some aspects, the nucleic acid sequence encoding the AAV vector A06049 includes SEQ ID NO: 179.

[0229] (Table 23) A06049:scAAV-2x_mU7prom-SCN1A1z15 / z6 -mU7term_mU1prom- SCN1A1z15 / z6 -mU1term; (Sheep loop without 5'ISD and eSL, short) (Equivalent to synthetic SR220592); Nucleotide sequence of plasmid element in order from N-terminus to C-terminus. TIFF2026536590000066.tif227166

[0230] The nucleotide sequence of A06049 (the entire transgene from ITR to ITR): TIFF2026536590000067.tif165160

[0231] Another exemplary AAV vector of this disclosure that targets SCN1A exon 20N is A06050. The elements of A06050 are shown in Table 24. In some aspects, the nucleic acid sequence encoding the AAV vector A06050 includes SEQ ID NO: 180.

[0232] (Table 24) A06050:scAAV-2x_mU7prom-SCN1A1z11 / z15-mU7term_mU1prom- SCN1A1z11 / z15-mU1term; (Sheep loop, short, lacking 5'ISD and SL) (Equivalent to synthetic SR220593); Nucleotide sequence of plasmid element from N-terminus to C-terminus. TIFF2026536590000068.tif219166

[0233] The nucleotide sequence of A06050 (the entire transgene from ITR to ITR): TIFF2026536590000069.tif164160

[0234] This specification provides SCN1A-targeting vector constructs comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to a nucleic acid sequence shown in any one of SEQ ID NO: 162 to 180. In some embodiments, the SCN1A-targeting vector construct comprises the nucleic acid sequence shown in SEQ ID NO: 162. In some embodiments, the SCN1A-targeting vector construct comprises the nucleic acid sequence shown in SEQ ID NO: 163. In some embodiments, the SCN1A-targeting vector construct comprises the nucleic acid sequence shown in SEQ ID NO: 164. In some embodiments, the SCN1A-targeting vector construct comprises the nucleic acid sequence shown in SEQ ID NO: 165. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 166. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 167. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 168. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 169. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 170. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 171. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 172. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 173. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 174. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 175.In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 176. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 177. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 178. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 179. In some embodiments, the vector construct targeting SCN1A includes the nucleic acid sequence shown in SEQ ID NO: 180.

[0235] nucleic acid The NOI (nucleotide sequence of interest) includes, but is not limited to, any nucleotide sequence or transgene to which the vector can deliver. The NOI may be synthetic, derived from native DNA or RNA, codon-optimized, recombinant RNA / DNA, cDNA, partial genomic DNA, and / or a combination thereof. The NOI may be a coding region or a partial coding region, or not a coding region. The NOI may be sense-oriented or antisense-oriented RNA / DNA. The NOI may be snRNA. The NOI is also referred to herein as a transgene, heterologous sequence, gene, or therapeutic gene, but is not limited to these terms. The NOI may also encode RNA (ribonucleoprotein complex), POI (protein of interest), a portion of a POI, a variant version of a POI, or a variant of a POI. The POI may be similar to or correspond to a wild-type protein. The POI may also be a fusion protein or a ribonucleoprotein complex, such as snRNP. In some respects, the RNA sequences disclosed herein may be represented as DNA sequences, and deriving RNA sequences from certain DNA sequences is within the capabilities of those skilled in the art. For example, the spacer sequences of this disclosure can represent the uracil base as either U or T. Those skilled in the art will readily understand that T or U can be used interchangeably in RNA sequences to represent uracil.

[0236] Codon optimization In some embodiments, the NOI or transgene, for example, the nucleic acid sequence of this disclosure, is a codon-optimized nucleic acid sequence.

[0237] Codon optimization is a well-known technique in the art. Codon optimization relates to the fact that different cells have different usage frequencies for certain codons. This codon bias corresponds to a bias where certain tRNAs are relatively abundant in certain cell types. It is possible to increase expression by modifying the codons in a sequence to match the relatively abundant corresponding tRNAs. It is also possible to decrease expression by intentionally selecting codons that are known to have low levels of corresponding tRNAs in certain cell types. Codon usage tables are known in the art for mammalian cells and for various other organisms. It is possible to construct nucleic acid sequences based on the genetic code. In some embodiments, such sequences are optimized for expression in host cells or target cells, for example, host cells used to express snRNA, or cells in which the methods of this disclosure are carried out (for example, mammalian cells, e.g., human cells). Using codon priority and codon usage tables for a particular species, it is possible to manipulate isolated nucleic acid molecules to encode snRNAs that take advantage of the codon usage priority of that particular species. In some embodiments, the isolated nucleic acid molecule (which may be part of the vector) comprises at least one coding sequence that is codon-optimized for expression in eukaryotic cells, or at least one coding sequence that is codon-optimized for expression in human cells. In one embodiment, such a codon-optimized coding sequence has sequence identity of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with respect to its corresponding wild-type sequence or original sequence.In another embodiment, a nucleic acid sequence optimized for eukaryotic cell codons encodes an snRNA having at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to its corresponding wild-type or original sequence. In another embodiment, various clones containing functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same sequence, can be constructed using conventional techniques. Silent mutations in coding sequences are due to degeneracy (i.e., redundancy) of the genetic code, where multiple codons can encode the same amino acid residue. Therefore, for example, leucine may be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine may be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine may be encoded by AAT or AAC; aspartic acid may be encoded by GAT or GAC; cysteine ​​may be encoded by TGT or TGC; alanine may be encoded by GCT, GCC, GCA, or GCG; glutamine may be encoded by CAA or CAG; tyrosine may be encoded by TAT or TAC; and isoleucine may be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3rd Edition, WH 5 Freeman and Co., NY).

[0238] In some embodiments, codon-optimized sequences exhibit at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased transcription or translation in human subjects compared to wild-type or non-codon-optimized nucleic acid sequences.

[0239] In some aspects, codon-optimized nucleic acid sequences exhibit increased stability. In some aspects, codon-optimized nucleic acid sequences exhibit increased stability due to increased resistance to hydrolysis. In some embodiments, codon-optimized sequences exhibit increased stability of at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% compared to wild-type or non-codon-optimized nucleic acid sequences. In some embodiments, codon-optimized sequences exhibit increased resistance to hydrolysis of at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% in human subjects compared to wild-type or non-codon-optimized nucleic acid sequences.

[0240] In some cases, a codon-optimized nucleic acid sequence does not need to contain donor splice sites. In some cases, a codon-optimized nucleic acid sequence may contain approximately 1 or fewer, or approximately 2 or fewer, or approximately 3 or fewer, or approximately 4 or fewer, or approximately 5 or fewer, or approximately 6 or fewer, or approximately 7 or fewer, or approximately 8 or fewer, or approximately 9 or fewer, or approximately 10 or fewer donor splice sites. In some cases, a codon-optimized nucleic acid sequence contains at least 1 fewer, or at least 2 fewer, or at least 3 fewer, or at least 4 fewer, or at least 5 fewer, or at least 6 fewer, or at least 7 fewer, or at least 8 fewer, or at least 9 fewer, or at least 10 fewer donor splice sites compared to a non-codon-optimized nucleic acid sequence.

[0241] While we do not wish to be bound by theory, removing donor splice sites in codon-optimized nucleic acid sequences prevents cryptic splicing, potentially leading to unexpected and unpredictable increases in the expression of proteins of interest in vivo. Furthermore, cryptic splicing can vary among different subjects, meaning that the expression levels of proteins containing donor splice sites can also vary unpredictably among different subjects. Such unpredictability is unacceptable in the context of human therapy. Therefore, codon-optimized nucleic acid sequences lacking donor splice sites unexpectedly and surprisingly enable increased protein expression in human subjects and homogenize protein expression across various human subjects.

[0242] In some aspects, codon-optimized nucleic acid sequences may have a different GC content than non-codon-optimized nucleic acid sequences. In some aspects, the GC content of codon-optimized nucleic acid sequences is more evenly distributed throughout the sequence compared to non-codon-optimized nucleic acid sequences.

[0243] While we do not wish to be bound by theory, codon-optimized nucleic acid sequences exhibit a more uniform melting temperature ("Tm") throughout the entire transcript by more evenly distributing the GC content across the entire nucleic acid sequence. This uniformity of melting temperature unexpectedly leads to increased expression of codon-optimized nucleic acids in human subjects, because there is less polymerase and / or ribosome arrest during transcription and / or translation of the nucleic acid sequence.

[0244] In some aspects, codon-optimized nucleic acid sequences may have fewer repressive microRNA target binding sites compared to non-codon-optimized nucleic acid sequences. In some aspects, codon-optimized nucleic acid sequences may have at least one fewer, or at least two fewer, or at least three fewer, or at least four fewer, or at least five fewer, or at least six fewer, or at least seven fewer, or at least eight fewer, or at least nine fewer, or at least ten fewer, or at least ten fewer, repressive microRNA target binding sites compared to non-codon-optimized nucleic acid sequences.

[0245] While we don't want to be constrained by theory, codon-optimized nucleic acid sequences with fewer repressive microRNA target binding sites unexpectedly show increased expression in human subjects.

[0246] Although not always explicitly stated, it should be understood that the sequences provided herein can be used to provide expression products and also to provide substantially identical sequences that encode RNA or are expressed to produce proteins having the same biological characteristics. These “biologically equivalent,” “biologically active,” or “equivalent” polypeptides are encoded by the equivalent polynucleotides described herein. They may have primary amino acid sequences that are at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to a reference polypeptide when compared using sequence identity methods operated under default conditions. Specific polypeptide sequences are provided as examples of certain embodiments. These include modifications to the sequence, such as replacing one amino acid with another amino acid having a similar charge. In addition, an equivalent polynucleotide is one that hybridizes to a reference polynucleotide or its complement under stringent conditions, or an equivalent polypeptide is a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide encoding a reference under stringent conditions, or by its complementary chain. Alternatively, an equivalent polypeptide or protein is one expressed from an equivalent polynucleotide.

[0247] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0248] Examples of stringent hybridization conditions include the following: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6x SSC to about 10x SSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include the following: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9x SSC to about 2x SSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5x SSC to about 2x SSC. Examples of highly stringent conditions include the following: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1x SSC, 0.1x SSC, or deionized water. Generally, the incubation time for hybridization is 5 minutes to 24 hours, which is accompanied by one, two, or more washing steps, and the incubation time for washing is about 1 minute, 2 minutes, or 15 minutes. SSC is a buffer of 0.15 M NaCl and 15 mM citrate. It is understood that equivalents of SSC using other buffer systems can also be utilized.

[0249] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the two molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the two sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

[0250] Cell Also provided herein are cells that comprise the RNA targeting systems described herein, and cells that comprise the expression constructs described herein. In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are prokaryotic cells.

[0251] In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are eukaryotic cells. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, mouse, feline, equine, porcine, canine, simian, or human cell. In some embodiments, the cell is a non-human mammalian cell, such as a non-human primate cell.

[0252] In some embodiments, the cells of the present disclosure are somatic cells. In some embodiments, the cells of the present disclosure are germ line cells. In some embodiments, the germ line cells of the present disclosure are not human cells.

[0253] In some embodiments of the compositions and methods of the Disclosure, the cells of the Disclosure are stem cells. In some embodiments, the cells of the Disclosure are embryonic stem cells. In some embodiments, the embryonic stem cells of the Disclosure are not human cells. In some embodiments, the cells of the Disclosure are multipotent or pluripotent stem cells. In some embodiments, the cells of the Disclosure are adult stem cells. In some embodiments, the cells of the Disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the cells of the Disclosure are hematopoietic stem cells (HSCs).

[0254] In some embodiments of the compositions and methods of the Disclosure, the somatic cells of the Disclosure are muscle cells. In some embodiments, the muscle cells of the Disclosure are myoblasts or muscle cells. In some embodiments, the muscle cells of the Disclosure are cardiomyocytes, skeletal muscle cells, or smooth muscle cells. In some embodiments, the muscle cells of the Disclosure are striated muscle cells. In one embodiment, single or more cells of a patient treated with the compositions disclosed in the specification include, but are not limited to, skeletal muscle (developing and mature muscle fibers, as well as satellite cells), neuromuscular junctions, cardiomyocytes, smooth muscle cells, peripheral nervous system (neurons), peripheral motor neurons, and / or sensory neurons.

[0255] In some embodiments of the compositions and methods disclosed herein, somatic cells of the disclosure are nerve cells. In one embodiment, single or more cells of a patient receiving treatment with the compositions disclosed herein include, but are not limited to, cells of the central nervous system (neurons), cells of the peripheral nervous system (neurons), peripheral motor neurons, sensory neurons, cortical interneurons, or GABAergic inhibitory interneurons. In one embodiment, nerve cells are glial cells.

[0256] In some embodiments of the compositions and methods of the Disclosure, the somatic cells of the Disclosure are fibroblasts or epithelial cells. In some embodiments, the epithelial cells of the Disclosure form squamous epithelium, cuboidal epithelium, columnar epithelium, stratified epithelium, pseudostratified columnar epithelium, or transitional epithelium. In some embodiments, the epithelial cells of the Disclosure form glands, which include, but are not limited to, the pineal gland, thymus, pituitary gland, thyroid gland, adrenal gland, apocrine gland, holocrine gland, merocrine gland, serous gland, mucous gland, and sebaceous gland. In some embodiments, the epithelial cells of the Disclosure are in contact with the outer surface of organs, which include, but are not limited to, the lungs, spleen, stomach, pancreas, bladder, intestine, kidney, gallbladder, liver, larynx, or pharynx. In some embodiments, the epithelial cells of the Disclosure are in contact with the outer surface of blood vessels or veins.

[0257] In some aspects of this disclosure, somatic cells are cells of the eye. Cells of the eye include, but are not limited to, corneal epithelial cells, keratinocytes, retinal pigment epithelial (RPE) cells, lens epithelial cells, iris pigment epithelial cells, conjunctival fibroblasts, ciliary body achromatophylline cells, trabecular meshwork cells, choroidal fibroblasts, and conjunctival epithelial cells. In some aspects, cells of the eye are retinal cells or corneal cells. In one aspect, retinal cells are photoreceptor cells or retinal pigment epithelial cells. In another aspect, retinal cells are ganglion cells, amacrine cells, bipolar cells, horizontal cells, Müller glial cells, rod cells, or cone cells. In some aspects of the compositions and methods of this disclosure, somatic cells of this disclosure are primary cells.

[0258] In some aspects of the compositions and methods of this disclosure, the somatic cells of this disclosure are cultured cells.

[0259] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are in vivo, in vitro, ex vivo, or in situ.

[0260] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are autologous or allogeneic.

[0261] How to use This disclosure provides a method for encoding RNA or expressing an NOI in cells using the snRNA system disclosed herein. In one embodiment, this disclosure provides a method for modifying the activity of RNA or a protein encoded by an RNA molecule, the method comprising the step of contacting a target RNA molecule with a composition of this disclosure under conditions suitable for binding to an RNA molecule.

[0262] This disclosure provides a method for modifying the expression level of an RNA molecule of the Disclosure or a protein encoded by such RNA molecule, the method comprising the step of contacting a cell containing the RNA molecule with a composition of the Disclosure under conditions suitable for binding to the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition of the Disclosure comprises a vector containing an snRNA sequence. In some embodiments, the vector is an AAV.

[0263] This disclosure provides a method for modifying the expression level of an RNA molecule of the Disclosure or a protein encoded by such RNA molecule, the method comprising the step of contacting the RNA molecule with a composition of the Disclosure under conditions suitable for knockdown, blocking, splicing, multi-targeting, frame recovery, or editing of the target RNA. In some embodiments, the composition of the Disclosure comprises a vector comprising an snRNA sequence. In some embodiments, the vector is an AAV.

[0264] This disclosure provides a method for modifying the activity of a target RNA or a protein encoded by a target RNA molecule, the method comprising the step of contacting a composition with a cell containing the RNA molecule under conditions suitable for knockdown, blocking, splicing, multi-targeting, frame recovery, or editing of the target RNA. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition comprises a vector containing an snRNA sequence disclosed herein. In some embodiments, the vector is an AAV.

[0265] This disclosure provides a method for treating a disease or disorder, the method comprising the step of administering a therapeutically effective amount of the snRNA composition of this disclosure to a target.

[0266] This disclosure provides a method for treating a disease in a patient requiring treatment, the method comprising the step of administering to the patient a therapeutically effective amount of the snRNA composition of this disclosure, wherein the composition comprises a vector comprising the snRNA sequence disclosed herein, wherein the composition restores the frame and function of a target RNA and increases the expression level of the targeted RNA (compared to the expression level of the targeted RNA treated with a non-targeting (NT) control or compared to an untreated RNA). In another embodiment, the increase is 1x or greater. In another embodiment, the increase is 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x. In another embodiment, the increase is 10x or greater. In another embodiment, the increase is 10x to 20x. In another embodiment, the increase is 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x. In another embodiment, the gene therapy compositions disclosed herein result in 20% to 100% recovery and / or function when administered to a patient. In one embodiment, the percentage of recovery and / or function is one of 20-99%, 25-99%, 50-99%, 80-99%, 90-99%, or 95-99%. In one embodiment, the percentage of recovery and / or function is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment of gene therapy, the sequences disclosed herein improve protein expression and function by promoting an increase in the expression level of the correct RNA transcript. In another embodiment, the percentage of upregulation is 1.5 times or more of the targeted RNA. In some embodiments, the targeted RNA is SCN1A. In some embodiments, the targeted RNA is SCN1A exon 20N. In some embodiments, the targeted RNA is SYNGAP1. In some embodiments, the targeted RNA is SYNGAP1 exon 11 or harmful exon 11.

[0267] The present disclosure further provides a method of treating a disease or disorder in a subject, the method comprising the step of administering an RNA-targeting nucleic acid molecule (i.e., the snRNA of the present disclosure) or an AAV vector comprising the snRNA of the present disclosure.

[0268] In some aspects, the disease or disorder is epilepsy. In some aspects, the epilepsy is predisposing epilepsy.

[0269] In some aspects, the disease or disorder is Dravet syndrome. In some aspects, the RNA-targeting nucleic acid molecule or AAV vector targets an RNA sequence encoding NaV1.1 (SCN1A). In some aspects, the RNA sequence encoding SCN1A comprises an intron sequence or an exon sequence. In some aspects, the exon sequence comprises exon 20N of SCN1A or a flanking region thereof.

[0270] In some aspects, the disease or disorder is SYNGAP1-related epilepsy. In some aspects, the RNA-targeting nucleic acid molecule or AAV vector targets an RNA sequence encoding Synaptic Ras GTPase-activating protein 1 (SYNGAP1). In some aspects, the RNA sequence encoding SYNGAP1 comprises an intron sequence or an exon sequence. In some aspects, the exon sequence comprises exon 11 or deleterious exon 11 of SYNGAP1, or flanking regions thereof.

[0271] In some embodiments of the methods of the present disclosure, the subject of the present disclosure has been diagnosed as having the disease to be treated. In some embodiments, the subject of the present disclosure exhibits at least one sign or symptom of the disease or disorder to be treated. In some embodiments, the subject of the present disclosure exhibits at least one sign or symptom of the disease.

[0272] In some embodiments of the methods of the disclosure, the subject of the disclosure is female. In some embodiments of the methods of the disclosure, the subject of the disclosure is male. In some embodiments, the subject of the disclosure has two chromosomes, XX or XY. In some embodiments, the subject of the disclosure has two chromosomes, XX or XY, and a third chromosome, which is either X or Y.

[0273] In some manner of this disclosure, the subject matter of this disclosure is newborns, infants, children, adults, elderly adults, or older adults. In some manner of this disclosure, the subject matter of this disclosure is at least 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days old, 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, 15 days old, 16 days old, 17 days old, 18 days old, 19 days old, 20 days old, 21 days old, 22 days old, 23 days old, 24 days old, 25 days old, 26 days old, 27 days old, 28 days old, 29 days old, 30 days old, or 31 days old. In some embodiments of the method of disclosure, the subject of the disclosure is at least 1 month old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, or 12 months old. In some embodiments of the method of disclosure, the subject of the disclosure is at least 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 15 years old, 20 years old, 25 years old, 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, 95 years old, 100 years old, or any age in between those ages or any fraction of an age in between those ages.

[0274] In some embodiments of the methods disclosed herein, the subject matter of the disclosure is mammals. In some embodiments, the subject matter of the disclosure is non-human mammals.

[0275] In some embodiments of the methods of this disclosure, the subject matter of this disclosure is human beings.

[0276] In some embodiments of the methods of the Disclosure, the therapeutically effective dose comprises a single dose of the composition of the Disclosure. In some embodiments, the therapeutically effective dose comprises at least one dose of the composition of the Disclosure. In some embodiments, the therapeutically effective dose comprises one or more doses of the composition of the Disclosure.

[0277] In some embodiments of the methods of this disclosure, a therapeutically effective dose eliminates the signs or symptoms of the disease or disorder. In some embodiments, a therapeutically effective dose reduces the severity of the signs or symptoms of the disease or disorder. Signs and symptoms of Dravet syndrome whose severity may be reduced or eliminated using the compositions described herein include seizures (e.g., focal seizures, generalized seizures, febrile seizures, or grand mal seizures), ataxia, hypotonia, and gait with flexed knees. In some embodiments, seizures are not adequately controlled with the use of standard therapeutic apologics (e.g., valproic acid, clobazam, topiramate, stiripentol, fenfluramine, and / or cannabidiol).

[0278] In some embodiments of the methods disclosed herein, a therapeutically effective dose eliminates a disease or disorder (e.g., Dravet syndrome).

[0279] In some embodiments of the methods of this disclosure, a therapeutically effective dose eliminates a disease or disorder (e.g., SYNGAP1-associated epilepsy).

[0280] In some aspects of the methods disclosed herein, a therapeutically effective dose prevents the onset of a disease or disorder. In some aspects, a therapeutically effective dose delays the onset of a disease or disorder. In some aspects, a therapeutically effective dose of the compositions described herein delays the onset of SYNGAP1-associated epilepsy or Dravet syndrome up to 2, 3, 4, 5, 10, 15, 20, or 25 years of age.

[0281] In some embodiments, the therapeutically effective dose improves the prognosis of the subject.

[0282] In some embodiments of the methods of the present disclosure, the composition of the present disclosure is administered to a subject by intracerebral administration. In some embodiments of the methods of the present disclosure, the composition of the present disclosure is administered to a subject by intraventricular injection. In some embodiments, the composition of the present disclosure is administered to a subject by an intrastriatal route. In some embodiments, the composition of the present disclosure is administered to a subject by stereotactic injection or infusion. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered to the brain. In some embodiments of the methods of the present disclosure, the composition of the present disclosure is administered topically to a subject.

[0283] In some embodiments, the compositions disclosed herein are formulated as pharmaceutical compositions. Briefly, the pharmaceutical compositions for the uses disclosed herein may, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, comprise proteins or polynucleotides encoding proteins, which are optionally contained in an AAV, and which AAV is also optionally immunoorthogonal. Such compositions may comprise: buffers, e.g., neutral buffered saline, phosphate-buffered saline, etc.; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for routes of administration such as oral, enteral, topical, transdermal, intranasal, and / or inhalation; and may also be formulated for routes of administration by injection or infusion, such as intravenous, intramuscular, subpia, intrathecal, intraparenchymal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and / or parenteral administration. In some embodiments, the compositions of this disclosure may be formulated for intracerebral or intrastriatal administration. [Examples]

[0284] The examples described herein are for illustrative purposes only and are not intended to limit the claimed invention.

[0285] Example 1: Elimination of harmful exon 20N in SCN1A material and method In vitro assay Skipping of harmful exons and quantification of productive SCN1A mRNA. Splicing assays to evaluate exon skipping were performed in HeLa or HepG2 cells transfected with plasmids expressing snRNA or synthetic snRNA. For SNC1A + / - GABAergic neurons, cells were transduced for 7 days using AAV9-2x snRNA cassettes (A05290 or A05291) of various MOIs. RNA was extracted by KingFisher using the Flex MirVana RNA extraction kit or the Zymo Direct-zol-96 MagBead RNA extraction kit. For exon skipping, cDNA was prepared using SuperScript IV RT. PCR was performed using KOD Xtreme hot-start polymerase and primers annealing to exons 20 and 21. 1 μL of PCR product was analyzed using a D1000 TapeStation. For productive SCN1A, qRT-PCR was performed using Ultraplex 1-Step Toughmix (QuantaBio) and primers and probes targeting the SCN1A inter-exon junction in exons 20 / 21. Custom primers and probes for GAPDH or ATP5b were used to quantify reference genes for normalization. Relative mRNA levels of SCN1A were calculated using the ΔΔCt method. For in vivo studies, WT mice received ICV injections of 5E10 vg / animal AAV9 A05290 or A05291 at P0. At P20, multiple areas of the cortex and hippocampus were collected and analyzed for snRNA expression, SCN1A exon 20N skipping, and productive SNC1A RNA and protein expression.

[0286] result In vitro evaluation of U7 snRNA targeting SCN1A exon 20N. U7 snRNA was engineered to bind to splicing regulatory sequences (acceptor and donor sites, or splicing enhancer sequences) within the SCN1A harmful exon 20N, promoting skipping and the restoration of frame and function (Figure 1A).

[0287] Figure 1B shows the SCN1A exon 20N non-skipping band (containing the harmful exon, upper fragment at 669 bp) or skipped product (exclusion of the harmful exon, lower band at 605 bp) in HeLa cells. The results are quantified in Figure 1C. Figure 2A shows tapestation images of RT-PCR products after treatment with U7 snRNA using a single and fused U7 spacer (cloned within a pcDNA plasmid), showing the band where SCN1A exon 20N is not skipped or the skipped product in HepG2 cells. The data are quantified in Figure 2B. The expression levels of productive (harmless exon 20N) SCN1A, determined by qRT-PCR after treatment with single and fused U7 snRNA, are shown in Figure 2C.

[0288] Conclusion: These data indicate that multiple U7 snRNA spacers (single and fused) promote skipping of harmful exon 20N and increase productive SCN1A mRNA expression in vitro. Fusion spacers exhibit more robust skipping in vitro.

[0289] Figure 3A shows the quantification of RT-PCR products as a percentage of harmful exon 20N inclusion after treatment with U7 snRNA in HepG2 cells using fusion synthetic U7 spacers z15 / z6 and z11 / z15, which have further modifications to the ISD and stem-loop. Similarly, Figure 3B shows the expression of productive (harmless exon 20N) SCN1A, determined by qRT-PCR after treatment with U7 snRNA in HepG2 cells transfected with synthetic U7 snRNA. The synthetic RNAs tested are as follows: SR220251: non-targeting; SR220516: targeting mod1; SR220519: non-targeting mod2; SR220522: non-targeting mod3; SR220346: z15 / z6; SR220517: z15 / z6 mod1; SR220520: z15 / z6 mod2; SR220523: z15 / z6 mod3; SR220347: z11 / z15; SR220518: z11 / z15 mod1; SR220521: z11 / z15 mod2; and SR220524: z11 / z15 mod3 (mod1 is without ISD, mod2 is natural mouse loop / without ISD, and mod3 is sheep loop / without ISD).

[0290] Conclusion: These data suggest that further optimization of snRNA ISD and loops increases the elimination of SCN1A harmful exon 20N in HepG2 cells.

[0291] Exon 20N skipping activity of U7 snRNA constructs in neurons of Dravet syndrome Based on the skipping activity of exon 20N in vitro, two optimal antisense U7 snRNA sequences were selected and packaged into a self-complementary AAV9 (scAAV9) genome, followed by AAV production. This snRNA packaging strategy minimizes repetitive sequences within the genome, resulting in high-titer, full-length self-complementary AAV preparations with the intended genome (including 2x snRNA), as confirmed by long-read sequencing (data not shown). The scAAV9 construct targeting the harmful exon was delivered to SCN1+ / - GABAergic inhibitory interneurons (in vitro) and also to P0 neonatal BL6 wild-type mice by intraventricular (ICV) injection (in vivo). In vitro, AAV9 null (empty capsid) cells and untreated (UNT) cells were used as negative controls.

[0292] Figure 4A shows tapestation images of RT-PCR products for detecting exon 20N skipping in SCN1A + / - GABAergic neurons and cells after treatment with U7 snRNA using AAV9 (a dual U7 snRNA cassette expressing fusion spacers under the mouse U7 promoter and mouse U1 promoter) while increasing MOI; A05290 (U7 fusion z15+z6) and A05291 (U7 fusion z11+z15).

[0293] The data are quantified in Figures 4B and 4C. Seven days after transduction with AAV9, complete elimination of SCN1A harmful exon 20N and a tenfold increase in productive SCN1A RNA occurred in SCN1A+ / - GABAergic neurons mediated by U7 snRNA (Figure 4C).

[0294] Conclusion: These data demonstrate the elimination of SCN1A harmful exons in SCN1A + / - GABAergic neurons 7 days after transduction using AAV9, induced by two different read U7 snRNA constructs.

[0295] The read constructs A05290 and A05291 (both containing a 2x U7 fusion spacer cassette) were further evaluated in vivo. The study design is shown in Figure 5A. Figure 5B shows representative tapestation images of RT-PCR products from cortical and hippocampal samples after treatment with U7 snRNA using A05290 and A05291, indicating bands where SCN1A harmful exon 21N (the mouse equivalent of harmful exon 20N in humans) was not skipped, or products where it was skipped. Vehicle-treated samples were used as negative controls. Data were quantified from different sections of cortex and hippocampus in Figure 5C. These data indicate that U7 snRNA delivered by scAAV9 can promote efficient skipping of the SCN1A harmful exon in the cortex and hippocampus of mice after neonatal ICV delivery.

[0296] Figure 5D shows the levels of productive (harmless exon-free) SCN1A mRNA in three cortical areas and two hippocampal areas 20 days after ICV injection of AAV9 expressing a dual U7 snRNA cassette. Figure 5E shows the expression levels of SCN1A / Nav1.1 protein in the cortex and hippocampus 20 days after ICV injection of AAV9 expressing a dual U7 snRNA cassette. Figure 5F shows snRNA expression in the cortex and hippocampus after treatment with snRNA-expressing AAV9. These data indicate that U7 snRNA delivered by scAAV9 increases productive SCN1A mRNA and protein in the cortex and hippocampus of mice after neonatal ICV delivery.

[0297] Conclusion: Dose-dependent expression of U7 snRNA and complete skipping of the harmful exon 20N were observed in neurons with Dravet syndrome 7 days after treatment. Similarly, complete exon skipping was observed in the cortical and hippocampal regions of mice, resulting in an approximately 2.0-fold increase in productive SCN1A mRNA and Nav1.1 protein 3 weeks after treatment. Overall, these data indicate that snRNA delivered by AAV can be used to successfully target the harmful SCN1A exon in vitro and in vivo to rescue haploinsufficiency.

[0298] Example 2: Elimination of harmful exon 11 of SYNGAP1 Inclusion of SYNGAP harmful exon 11 (shown as a shaded square), which contains an immature stop codon (PTC), leads to transcript degradation and haploinsufficiency via the nonsense mutation-dependent degradation mechanism (NMD) (Figure 6A). Figure 6B illustrates the mechanism of action of U7 snRNA skipping SYNGAP harmful exon 11 to restore frame and function. U7 snRNA was engineered to bind to a splicing regulatory sequence within SYNGAP1 harmful exon 11 to promote skipping and restoration of frame and function. Figure 6C shows representative tapestation images of RT-PCR products after treatment with U7 snRNA using individual U7 spacers (in pcDNA, z22, z23, and z24), showing bands where SYNGAP harmful exon 11 is not skipped (containing the harmful exon, upper fragment) or products where it is skipped (excluded, lower band). Non-targeting U7(NT) was used as a negative control. ASO, which has been previously shown to promote the elimination of harmful exon 11, was used as a positive control. (Figure 6D) shows the quantitative results of tapestation in Hek293-T cells, plotted as the percentage of harmful exon elimination after treatment with U7 snRNA of multiple single and fusion spacers targeting distinct areas of SYNGAP1 premRNA (graph and schematic diagram below are shown).

[0299] Conclusion: U7 snRNA can bind to multiple regions of SYNGAP1 premRNA and can promote the skipping of harmful exon 11 in HEK293T cells.

[0300] Quantification of endogenous SYNGAP1 harmful exon 11 skipping as a percentage determined by splicing assay after treatment with monosynthetic U7 snRNA (Figure 7A), or quantification of productive SYNGAP1 mRNA expression as determined by qRT-PCR (Figure 7B), was performed in HEK293-T cells. Non-targeting U7(NT) was used as a negative control. Quantification of endogenous SYNGAP1 harmful exon 11 skipping as a percentage determined by splicing assay (Figure 7C), or quantification of productive SYNGAP1 mRNA expression as determined by qRT-PCR (Figure 7D), was performed in HEK293-T cells after treatment with novel optimized monosynthetic and fusion U7 snRNAs. Non-targeting U7(NT) was used as a negative control.

[0301] Conclusion: Optimized, single and fusion synthetic U7 snRNAs promote efficient skipping of SYNGAP1 harmful exon 11 and increase the expression of productive SYNGAP1 mRNA in HEK293T cells.

[0302] Figure 8A shows a tapestation image of RT-PCR products as levels of SYNGAP1 exon 11 in neural progenitor cells (NPCs) from a patient containing the R1240X mutation. The patient's NPCs show high levels of harmful SYNGAP1 exon 11 (upper band). Quantification of RT-PCR products as percentage of SYNGAP1 harmful exon 11 skipping after treatment with U7 snRNA in patient-derived NPCs (Figure 8B). Non-targeting U7 (NT) was used as a negative control. Figure 8C shows the expression of productive (harmless exon 11) SYNGAP1 mRNA as determined by qRT-PCR after treatment with U7 snRNA.

[0303] Conclusion: Multiple U7 snRNAs promote efficient skipping of harmful exon 11 of SYNGAP1 and increase the expression of productive SYNGAP1 mRNA in patient-derived NPCs.

[0304] Inclusion by reference All documents cited herein, including any cross-referenced patents or applications, or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. No citation of any document constitutes prior art to any invention disclosed or embodied herein, nor does such citation alone or in combination with any other single or multiple references constitute teaching, suggestion, or disclosure of any such invention. Furthermore, if any meaning or definition of a term herein is inconsistent with any meaning or definition of the same term in any document incorporated by reference, the meaning or definition assigned to the term herein shall apply.

[0305] Other embodiments While certain aspects of this disclosure are illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of this disclosure. The attached claims include all such changes and modifications that fall within the scope of this disclosure.

Claims

1. An RNA-targeting nucleic acid molecule comprising nuclear small RNA (snRNA), wherein the snRNA contains a targeting sequence that binds to the SYNGAP1 RNA sequence.

2. The RNA targeting nucleic acid molecule according to claim 1, wherein the SYNGAP1 RNA sequence is exon 11.

3. The RNA targeting nucleic acid molecule according to claim 1, wherein the SYNGAP1 targeting sequence comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 181–279.

4. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA includes a stem-loop (SL).

5. The RNA targeting nucleic acid molecule according to claim 4, wherein the SL comprises one or more nucleic acid sequences represented by any one of SEQ ID NO: 84 to 124.

6. The RNA targeting nucleic acid molecule according to claim 1, wherein the SYNGAP1 RNA sequence is a premRNA sequence or an mRNA sequence.

7. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA includes an Sm-binding domain (SmBD).

8. The RNA targeting nucleic acid molecule according to claim 7, wherein the SmBD is U1, U2, U4, or U5 SmBD.

9. The RNA targeting nucleic acid molecule according to claim 7, wherein the SmBD comprises a nucleic acid sequence represented by either SEQ ID NO: 160 or 161.

10. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA includes a 5' interaction stabilization domain (5'ISD).

11. The RNA targeting nucleic acid molecule according to claim 10, wherein the 5'ISD comprises the nucleotide sequence ggagt, cctct, ggaggt, cctcct, agccag, ggaag, gaagaag, gttg, ccgaa, taaggag, gaag, or ggctt.

12. A vector comprising one or more snRNAs according to claim 1.

13. The vector according to claim 14, which is an AAV vector.

14. The AAV vector according to claim 13, wherein the snRNA is functionally linked to the promoter.

15. The AAV vector according to claim 13, wherein the snRNA is functionally linked to a U7 promoter or a U1 promoter.

16. The AAV vector according to claim 13, wherein the snRNA is functionally linked to a downstream terminator (DT).

17. The AAV vector according to claim 13, wherein the snRNA is functionally ligated to a downstream terminator of U7 or a downstream terminator of U1.

18. The AAV vector according to claim 13, wherein the vector comprises at least one, at least two, at least three, at least four, or at least five snRNAs.

19. The AAV vector according to claim 18, wherein each of the at least one, at least two, at least three, at least four, or at least five snRNAs targets the same target RNA sequence.

20. The AAV vector according to claim 18, wherein each snRNA is separated by a buffer sequence.

21. The AAV vector according to claim 20, wherein the buffer sequence comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence shown in any one of SEQ ID NO: 151 to 157.

22. A method for targeting one or more target RNAs of interest and performing exon skipping of said one or more target RNAs, comprising the step of bringing the snRNA described in claim 1 into contact with a cell containing said one or more target RNAs.

23. A SYNGAP1 RNA targeting nucleic acid molecule containing a targeting sequence indicated by one of the following SEQ ID NOs: 181–279.

24. A method for treating a disease or disorder in a subject, comprising the step of administering to the subject an RNA targeting nucleic acid molecule according to claim 1 or an AAV vector according to claim 13.

25. The method according to claim 24, wherein the disease or disorder is predisposed epilepsy.

26. The method according to claim 24, wherein the disease or disorder is SYNGAP1-associated epilepsy.

27. The method according to claim 24, wherein the administration is systemic, intravenous, or intraventricular.

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