Therapeutic regulation of SCN2a splicing

Abstract

Disclosed herein are systems for modifying nucleic acid splicing of a target RNA such as a SCN2A mRNA. The system may be useful in a method such as a method for treating a neurodevelopmental disorder. The system may include an expression system, or a modified small nuclear RNA (snRNA) that includes an exonic splicing silencer (ESS) sequence, and a target nucleic acid sequence that targets the target RNA.

Description

[0001] Attorney Docket No. 062692-509001 WO

[0002] THERAPEUTIC REGULATION OF SCN2A SPLICING

[0003] CROSS-REFERENCE

[0004] [1] This application claims the benefit of U.S. Provisional Application No. 63 / 691,080, filed September 5, 2024, U.S. Provisional Application No. 63 / 705,773, filed October 10, 2024, and U.S. Provisional Application No. 63 / 786,641, filed April 10, 2025, which applications are incorporated herein by reference.

[0005] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0006] [2] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 062692-50900 IWO.xml, created August 26, 2025, which is 1,472,407 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] [3] Neurodevelopmental disorders are a concern for many individuals, and improved treatments are needed.

[0009] SUMMARY

[0010] [4] Disclosed herein are systems for modifying nucleic acid splicing. The system may affect splicing and expression of sodium channel protein type 2 subunit alpha (SCN2A). The system may be useful in a method such as a method for treating of a neurodevelopmental disorder. The disclosure herein helps address unmet needs for improved, safe and effective treatments for neurodevelopmental disorders.

[0011] [5] Disclosed herein, in some embodiments, is a system for modifying nucleic acid splicing, comprising: an engineered U7 small nuclear RNA (snRNA) comprising a targeting nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the engineered U7 snRNA further comprises an exonic splicing silencer (ESS) nucleic acid sequence. In some embodiments, the engineered U7 snRNA does not comprise an ESS nucleic acid sequence. Disclosed herein, in some embodiments, is a system modifying nucleic acid splicing, comprised of an exonic splicing silencer (ESS) nucleic acid sequence; and a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the alternatively spliced region comprises a stop codon. In some embodiments, the SCN2A comprises a human SCN2A. In some embodiments, the alternatively spliced region is an alternatively spliced exon. In some embodiments, the alternatively spliced exon comprises exon 13N. In some embodiments, the exon 13N comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2. In some Attorney Docket No. 062692-509001 WO embodiments, the ESS recruits a protein factor or group of factors that reduce or silence splicing of the RNA encoding SCN2A. In some embodiments, the ESS nucleic acid sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an ESS nucleic acid sequence in Table 2. In some embodiments, the ESS nucleic acid sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an ESS nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence is 10-60 nucleotides in length. In some embodiments, the target nucleic acid sequence binds to the alternatively spliced region. In some embodiments, the targeting nucleic acid sequence is fully reverse complementary or partially reverse complementary to the targeted region. In some embodiments, the targeting nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reverse complementary to the targeted region. In some embodiments, the portion of the alternatively spliced region is within a 5’ half or 5’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 50 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence is a human variant of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence is a human variant of a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence is 3’ or downstream relative to the ESS nucleic acid sequence. In some embodiments, the nucleic acid sequence further comprises a Sm binding site. In some embodiments, the Sm binding site comprises AAUUUGUCUAG (SEQ ID NO: 112) or AAUUUUUGGAG (SEQ ID NO: 113; smOPT). In some embodiments, the Sm binding site is 3’ or downstream relative to the ESS nucleic acid sequence or target nucleic acid sequence. In some embodiments, the nucleic acid sequence further comprises a hairpin sequence. In some embodiments, the hairpin sequence comprises a U7 small nuclear RNA (snRNA) hairpin sequence. In some embodiments, the U7 snRNA hairpin sequence comprises the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the hairpin sequence comprises a 3’ hairpin sequence. In some embodiments, the hairpin sequence is 3’ or downstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, or the Sm binding site. In some embodiments, the system comprises one or more RNA molecules. In some embodiments, the RNA is a modified U7 snRNA. In some embodiments, the system comprises a deoxyribonucleic acid (DNA). In some embodiments, the system comprises an expression cassette. In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, the promoter sequence comprises a mouse or human promoter sequence. In some embodiments, the promoter Attorney Docket No. 062692-509001 WO sequence comprises a U7 snRNA promoter sequence. In some embodiments, the promoter sequence comprises a U1 promoter sequence. In some embodiments, the promoter sequence comprises a mouse U7 snRNA (“Mm U7”) promoter sequence, a human U7 snRNA (“Hs U7”) promoter sequence, a mouse Ulal (“mulal” or “Mm Ulal”) promoter sequence, or a human Ul-1 (“HUI” or “Hs Ul-1”) promoter sequence, or a fragment or combination of fragments thereof. In some embodiments, the promoter sequence comprises a U7 snRNA promoter sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 or Ulal promoter sequence. In some embodiments, the promoter sequence comprises a mouse U7 promoter sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 or Ulal promoter sequence. In some embodiments, the promoter sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence is 5’ or upstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, the Sm binding site, or the hairpin sequence. In some embodiments, the nucleic acid system further comprises a terminator sequence. In some embodiments, the terminator sequence comprises mouse or human terminator sequence. In some embodiments, the terminator sequence comprises a U7 snRNA terminator sequence. In some embodiments, the terminator sequence comprises a U1 terminator sequence. In some embodiments, the terminator sequence comprises a Mm U7 terminator sequence, a Hs U7 terminator sequence, a mulal terminator sequence, or a HU 1 terminator sequence, or a fragment or combination of fragments thereof. In some embodiments, the terminator sequence comprises a U7 snRNA terminator sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 or Ulal terminator sequence. In some embodiments, the terminator sequence comprises a mouse U7 snRNA terminator sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 or Ulal terminator sequence. In some embodiments, the terminator sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence comprises a 3’ terminator sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the promoter sequence, the ESS nucleic acid sequence, the target nucleic acid sequence, the Sm binding site, or the hairpin sequence. In some embodiments, the components are combined together within a single nucleic acid. In some embodiments, the components are separated among multiple nucleic acids. In some embodiments, a pharmaceutical composition is described that contains the system and a pharmaceutically acceptable carrier. In some embodiments, a virus comprises the system. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, a cell comprises the system. In some embodiments, the cell is a neural cell. In some embodiments, a method comprising the pharmaceutical composition or virus is described. Described herein, in some embodiments, is a method of modifying splicing, comprising contacting a pre-mRNA encoding SCN2A with a recombinant nucleic acid sequence (e.g., a Attorney Docket No. 062692-509001 WO recombinant U7 snRNA sequence) that induces exclusion of exon 13N from a mature mRNA generated by the pre-mRNA. In some embodiments, the pre-mRNA is in a cell. In some embodiments, the cell is in a subject. In some embodiments, the subject has or is at risk of having a genetic disorder. In some embodiments, the subject has or is at risk of having a neurode velopmental disorder. In some embodiments, the neurodevelopmental disorder comprises intellectual disability, autistic features, or epilepsy. In some embodiments, the subject has or is at risk of having epilepsy.

[0012] [6] Described herein, in some embodiments, is a method comprising administering to the subject a composition that silences or reduces said splicing, the composition comprising a system, virus, or composition described herein. The composition may include a recombinant U7 snRNA sequence. In some embodiments, the method increases an amount of a productive isoform of SCN2A, relative to a control or a baseline amount of said productive isoform. In some embodiments, the method increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a control or baseline amount of the NaV1.2 channel or protein. In some embodiments, the method improves sodium transport, relative to a control or baseline amount. In some embodiments, the method increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a control or baseline amount of the NaV1.2 channel or protein measured within 1, 2, 3, or 4 weeks postadministration. In some embodiments, the method increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a control or baseline amount of the NaV1.2 channel or protein measured at least 1, 2, 3, or 4 weeks post-administration. In some embodiments, the method improves sodium transport, relative to a control or baseline amount measured within 1, 2, 3, or 4 weeks post-administration. In some embodiments, the method improves sodium transport, relative to a control or baseline amount measured at least 1, 2, 3, or 4 weeks post-administration.

[0013] [7] Described herein, in some embodiments, is a method of treating or preventing a neurodevelopmental disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant U7 small nuclear RNA (snRNA) composition that silences or reduces splicing of an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the neurodevelopmental disorder comprises intellectual disability, autistic features, or epilepsy. A composition such as a synthetic composition may include a recombinant U7 snRNA sequence. In some embodiments, the alternatively spliced region comprises exon 13N of the RNA. In some embodiments, the administration increases an amount of a productive isoform of SCN2A in the subject, relative to a control or baseline amount of said productive isoform. In some embodiments, the administration increases an amount of a NaV1.2 channel or protein (e.g. SCN2A protein) in the subject, relative to a control or baseline amount of the NaV1.2 channel or protein. In some embodiments, the administration improves sodium transport in the subject, relative to a control or baseline amount. In some embodiments, the administration prevents the subject from having seizures, or reduces an amount or severity of the seizures of the subject relative to a baseline amount or severity. Attorney Docket No. 062692-509001 WO

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] [8] FIG. 1 is an image of a blot showing results of end-point RT-PCR with RNA from Neuro-2a cells. These results demonstrate increased levels of exon 13N inclusion (top band) in the presence of cycloheximide (CHX), an inhibitor of nonsense-mediated decay (NMD).

[0016] [9] FIG. 2 is a pairwise alignment of mouse SCN2A and human SCN2A genomic regions including exon 13N, a non-productive exon whose inclusion results in nonsense-mediated decay (NMD).

[0017]

[0010] FIG. 3 is a plot showing results of U7 screening with a mouse U7 library and luciferase reporter encoding the differentially spliced Scn2a region upstream of luciferase. The y-axis is luciferase activity normalized to a mean of measurements for negative controls, Scramble-01 and Scramble-02. The dashed line in FIG. 3 graphically indicates an increase in reporter expression by 50%. The x-axis has numbers from 1 (on the left) to 96 (on the right), indicative of constructs that were tested, with values shown in Table 2. The circles on the extreme left, above “1” and “2” of the x-axis are data for control constructs (termed Scramble-01 and Scramble-02 in Table 2, respectively). The circles above “3” to “96” of the x-axis are data for test constructs, though not in the same order as Table 2.

[0018]

[0011] FIG. 4 shows results of an analysis of relative NMD transcript levels from Neuro-2a cells transfected with constructs expressing U7 cassettes containing single targeting sequences complementary to regions at both the 5’ and 3’ ends of mouse Scn2a exon 13N.

[0019]

[0012] FIG. 5 includes results of a protein analysis of NaV1.2 expression in ReNcell CX cultures transduced with AAVs expressing a scramble U7 or candidates 1, 2, or 9.

[0020]

[0013] FIG. 6 is a plot showing results of U7 screening with a human U7 library and a luciferase reporter encoding the mouse Scn2a region of differential splicing. The y-axis is luciferase activity normalized to a mean of measurements for negative controls, Scramble-01 and Scramble-03. The dotted line in FIG. 6 graphically indicates an increase in reporter expression by 50%. The x-axis has numbers from 1 (on the left) to 96 (on the right), indicative of constructs that were tested. The x-axis represents each construct tested, with values shown in Table 4. The circles at positions corresponding to 1 and 7 of the x-axis are data for control constructs (termed Scramble-01 and Scramble-03 in Table 4, respectively). The circles at positions corresponding to 2-6 and 8-96 of the x-axis are data for test constructs.

[0021]

[0014] FIG. 7 includes a schematic of a luciferase reporter containing a ubiquitous promoter (here, Ubc-SV40pA), a Kozak sequence, a fragment of mouse Scn2a exon 13, a fragment of mouse Scn2a intron 13, mouse Scn2a exon 13N, another fragment of mouse Scn2a intron 13, and a fragment of mouse Scn2a exon 14 fused in-frame to firefly luciferase.

[0022]

[0015] FIG. 8 is a bar graph showing results of U7 expression of the promoter / terminator pair hRNU12 / hRNUl-2. The x-axis is luciferase activity normalized to a mean of measurements for the Attorney Docket No. 062692-509001 WO full-length hRNUl-2 terminator (hRNUl-2_500). The dotted line in FIG. 8 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying hRNUl-2 terminator (100bp-500bp) lengths and Scramble as the negative control.

[0023]

[0016] FIG. 9 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu2-10 / hRNU12. The x-axis is luciferase activity normalized to a mean of measurements for the full-length hRNU12 terminator (hRNU12_500). The dotted line in FIG. 9 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying hRNU12 terminator (100bp-500bp) lengths and Scramble as the negative control.

[0024]

[0017] FIG. 10 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu2-10 / mRnu5g. The x-axis is luciferase activity normalized to a mean of measurements for the full-length mRnu5g terminator (mRnu5g _500). The dotted line in FIG. 10 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnu5g terminator (100bp-500bp) lengths and Scramble as the negative control.

[0025]

[0018] FIG. 11 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu3bl / hRNU5A-l. The x-axis is luciferase activity normalized to a mean of measurements for the full-length hRNU5A-l terminator (hRNU5A-l _500). The dotted line in FIG. 11 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying hRNU5A-l terminator (100bp-500bp) lengths and Scramble as the negative control.

[0026]

[0019] FIG. 12 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu2-10 / mRnulal. The x-axis is luciferase activity normalized to a mean of measurements for the full-length mRnu lai terminator (mRnulal_500). The dotted line in FIG. 12 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnulal terminator (100bp-500bp) lengths and Scramble as the negative control.

[0027]

[0020] FIG. 13 is a bar graph showing results of U7 expression of the promoter / terminator pair hRNU12 / mRnu7. The x-axis is luciferase activity normalized to a mean of measurements for the full- length mRnu7 terminator (mRnu7_500). The dotted line in FIG. 13 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnu7 terminator (100bp-500bp) lengths and Scramble as the negative control.

[0028]

[0021] FIG. 14 is a bar graph showing results of U7 expression of the promoter / terminator pair hRNU12 / hRNUl-2. The x-axis is luciferase activity normalized to a mean of measurements for the full-length hRNU12 promoter (hRNU12_500). The dotted line in FIG. 14 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying hRNU12 promoter (100bp-500bp) lengths and Scramble as the negative control. Attorney Docket No. 062692-509001 WO

[0029]

[0022] FIG. 15 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu2-10 / hRNU12. The x-axis is luciferase activity normalized to a mean of measurements for the full-length mRnu2-10 promoter (mRnu2-10_500). The dotted line in FIG. 15 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnu2-10 promoter (100bp-500bp) lengths and Scramble as the negative control.

[0030]

[0023] FIG. 16 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnu3bl / hRNU5A-l. The x-axis is luciferase activity normalized to a mean of measurements for the full-length mRnu3bl promoter (mRnu3bl_500 in the figure). The dotted line in FIG. 16 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnu3bl promoter lengths (100bp-500bp) and Scramble as the negative control.

[0031]

[0024] FIG. 17 is a bar graph showing results of U7 expression of the promoter / terminator pair mRnulal / mRnul l. The x-axis is luciferase activity normalized to a mean of measurements for the full-length mRnulal promoter (mRnulal_500). The dotted line in FIG. 17 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying mRnulal promoter (100bp-500bp) lengths and Scramble as the negative control.

[0032]

[0025] FIG. 18 is a bar graph showing results of U7 expression of the promoter / terminator pair hRNUl-2 / mRnu7. The x-axis is luciferase activity normalized to a mean of measurements for the full- length hRNUl-2 promoter (hRNUl-2_500). The dotted line in FIG. 18 graphically indicates the normal activity of the full-length promoter / terminator pair. The y-axis represents each construct tested, with varying hRNUl-2 promoter (100bp-500bp) lengths and Scramble as the negative control.

[0033]

[0026] FIG. 19 shows a bar graph of the relative NMD transcript levels from Neuro-2a cells transfected with U7 expressing constructs. The y-axis represents the fold change normalized to the negative control (Scramble). The x-axis represents each construct tested. The full-length and 400bp mRnu2-10 promoter (P400 or P500) were combined with PSE, DSE, or 3’ Box duplications. For each pair of bars, the left depicts Scn2a NMD isoform levels, while the right represents productive Scn2a isoform levels.

[0034]

[0027] FIG. 20 shows a bar graph of the relative NMD transcript levels from Neuro-2a cells transfected with U7 expressing constructs. The y-axis represents the fold change normalized to the full-length promoter / terminator pair (P500). The full-length and 400bp mRnu2-10 promoter (P400 or P500) were combined with PSE, DSE, or 3’ Box duplications. Constructs were also tested with or without (-K) Kozak- ATG motif in the terminator. For each pair of bars, the left depicts productive Scn2a isoform levels, while the right represents Scn2a NMD isoform levels.

[0035]

[0028] FIG. 21 is a plot showing relative normalized Nav1.2 protein levels from whole-brain lysates of Scn2a+ / mice treated with either saline or AAV-PHP.eB-ASl (3el3vg / kg) at postnatal day 14. Tissue was collected 4 weeks post-injection, analyzed via capillary-based protein analysis, and normalized to vinculin. Attorney Docket No. 062692-509001 WO

[0036] DETAILED DESCRIPTION

[0037]

[0029] Mutation of SCN2A, which encodes sodium channel NaV 1.2, is a leading genetic cause of neurode velopmental disorders. While gain-of-function mutations result in epileptic encephalopathy, SCN2A haploinsufficiency causes a distinct disorder characterized by intellectual disability, autistic features, and seizures. There are currently no disease-modifying therapies available to treat the core symptoms of SCN2A haploinsufficiency, but recovery of NaV1.2 to wild-type levels may provide a therapeutic benefit. Analysis of large-scale RNA sequencing data identified an alternate isoform of SCN2A that includes an extra exon, referred to as exon 13N. This alternate isoform is out of frame and results in nonsense-mediated decay (NMD).

[0038]

[0030] Precise pre-mRNA splicing may be important for appropriate protein translation and may depend on the presence of consensus ‘cis’ sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery. Point mutations at consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of a mutated gene. Splicing mutations may occur in introns or exons, and disrupt existing splice sites or splicing regulatory sequences (e.g., intronic and exonic silencers), create new ones, or activate cryptic ones. Such mutations may result in errors during splicing or improper intron removal, and cause alterations of an open reading frame (ORF). Alterations in ORFs may lead to truncated or extended mRNA products resulting in translated proteins being unable to perform normal functions due to misfolding or degradation. Loss or reduced expression of proteins due to mRNA spicing errors can lead to neurode velopmental disorders.

[0039]

[0031] Described herein are recombinant regulatory elements for expression systems. Also described are nucleic acid expression systems with recombinant regulatory elements. The regulatory elements and systems described herein address needs for improved expression systems, such as for gene editing or for transgenic expression.

[0040]

[0032] Described herein are compositions and methods for treating a neurodevelopmental disorder in a subject in need thereof. The compositions and methods described herein address an unmet need for safe and effective treatment of neurodevelopmental disorders. The neurodevelopmental disorder may be genetically caused, such as by a mutation in SCN2A (e.g., resulting in SCN2A haploinsufficiency). The mutation may lead to aberrant SCN2A splicing, or result in inclusion of exon 13N in a mature SCN2A mRNA transcript. Examples of neurodevelopmental disorders may include intellectual disability, autistic features, or epilepsy (e.g., including seizures).

[0041]

[0033] Some embodiments include a system for modifying nucleic acid splicing. The system may include an exonic splicing silencer (ESS) nucleic acid sequence. The system may include a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA). The target nucleic acid sequence may encode the sodium channel protein type 2 subunit alpha (SCN2A). Described herein, in some embodiments, is a system for modifying nucleic acid splicing comprising Attorney Docket No. 062692-509001 WO an exonic splicing silencer (ESS) nucleic acid sequence and a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A).

[0042]

[0034] Some embodiments relate to a system for modifying nucleic acid splicing, comprising: an engineered U7 small nuclear RNA (snRNA) comprising a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the engineered U7 snRNA further comprises an exonic splicing silencer (ESS) nucleic acid sequence. In some embodiments, the engineered U7 snRNA does not comprise an ESS nucleic acid sequence.

[0043]

[0035] Disclosed herein, in some embodiments, are systems for modifying nucleic acid splicing. In some embodiments, the system may include an exonic splicing silencer (ESS) nucleic acid sequence. In some embodiments, the system may include a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA). In some embodiments, the target nucleic acid sequence may encode the sodium channel protein type 2 subunit alpha (SCN2A).

[0044]

[0036] Described herein, in some embodiments, is a nucleic acid system that may include deoxyribonucleic acid (DNA). The DNA may include an expression cassette. In some embodiments, the nucleic acid system that may include ribonucleic acid (RNA). The RNA may be encoded by the expression cassette.

[0045] Target RNAs and Alternatively Spliced Regions of Target RNAs

[0046]

[0037] Described herein, in some embodiments, are methods or systems that affect splicing of a target RNA such as an SCN2A RNA. The RNA may be an mRNA (e.g. pre-mRNA). For example, the target RNA includes an SCN2A mRNA. In some embodiments, target RNA may include an SCN2A pre-mRNA. In some embodiments, target RNA includes a mature mRNA such as a mature SCN2A mRNA. the In some embodiments, the SCN2A comprises a mammalian SCN2A. In some embodiments, the SCN2A comprises a primate SCN2A. In some embodiments, the SCN2A comprises a human SCN2A. In some embodiments, the SCN2A comprises a rodent or mouse SCN2A.

[0047]

[0038] Some embodiments refer to or include an alternatively spliced region of a target RNA, such as an alternatively spliced region of an SCN2A RNA. For example, some embodiments of a system or method include or refer to a target nucleic acid sequence that targets an alternatively spliced region of a target RNA such as an SCN2A RNA.

[0048]

[0039] The alternatively spliced region may be or include an alternatively spliced exon. The alternatively spliced region may be out of frame with a productive transcript. This may lead to production of a downstream stop codon. In some embodiments, inclusion of the alternatively spliced region results in an mRNA (e.g., a mature SCN2A mRNA) having a premature stop codon. Inclusion of a premature stop codon may result in a non-productive mRNA. Inclusion of a premature stop codon may result in the mRNA being non-productive for functional or full-length SCN2A. Inclusion of a Attorney Docket No. 062692-509001 WO premature stop codon may result in nonsense mediated decay. In some embodiments, the alternatively spliced region is out of frame with an exon of a productive transcript. In some embodiments, the alternatively spliced region may include a stop codon. In some embodiments, the alternatively spliced region does not include a stop codon.

[0049]

[0040] In some embodiments, the alternatively spliced region may be an alternatively spliced exon. In some embodiments, the alternatively spliced region may not be an alternatively spliced exon. In some embodiments, the alternatively spliced region is an alternatively spliced intron. In some embodiments, the alternatively spliced exon comprises exon 13N (e.g. exon 13N of a mammalian SCN2A mRNA). In some embodiments, the alternatively spliced exon comprises exon 13N of a human SCN2A mRNA. In some embodiments, the alternatively spliced exon comprises exon 13N of a mouse SCN2A mRNA.

[0050]

[0041] A targeted region may include SEQ ID NO: 766. A targeted region may include part of SEQ ID NO: 766. A targeted region may include a sequence at least 99% identical to SEQ ID NO:

[0051] 766 or at least 99% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 98% identical to SEQ ID NO: 766 or at least 98% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 97% identical to SEQ ID NO: 766 or at least 97% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 96% identical to SEQ ID NO: 766 or at least 96% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 95% identical to SEQ ID NO: 766 or at least 95% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 94% identical to SEQ ID NO: 766 or at least 94% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 93% identical to SEQ ID NO: 766 or at least 93% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 92% identical to SEQ ID NO: 766 or at least 92% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 91% identical to SEQ ID NO: 766 or at least 91% identical to part of SEQ ID NO: 766. A targeted region may include a sequence at least 90% identical to SEQ ID NO: 766 or at least 90% identical to part of SEQ ID NO: 766.

[0052]

[0042] A targeted region may include SEQ ID NO: 767. A targeted region may include part of SEQ ID NO: 767. A targeted region may include a sequence at least 99% identical to SEQ ID NO:

[0053] 767 or at least 99% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 98% identical to SEQ ID NO: 767 or at least 98% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 97% identical to SEQ ID NO: 767 or at least 97% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 96% identical to SEQ ID NO: 767 or at least 96% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 95% identical to SEQ ID NO: 767 or at least 95% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 94% identical to SEQ ID NO: 767 or at least 94% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least Attorney Docket No. 062692-509001 WO

[0054] 93% identical to SEQ ID NO: 767 or at least 93% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 92% identical to SEQ ID NO: 767 or at least 92% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 91% identical to SEQ ID NO: 767 or at least 91% identical to part of SEQ ID NO: 767. A targeted region may include a sequence at least 90% identical to SEQ ID NO: 767 or at least 90% identical to part of SEQ ID NO: 767.

[0055]

[0043] In some embodiments, the exon may include a nucleic acid sequence identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 99% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 98% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 97% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 96% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 94% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 93% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 92% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 91% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 85% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 80% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence at least 75% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence of at least 70% identical to SEQ ID NO: 1. In some embodiments, the exon may include a nucleic acid sequence identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 99% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 98% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 97% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 96% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 95% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 94% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 93% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 92% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 91% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 90% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 85% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence at least 80% identical to SEQ ID NO: 2. Attorney Docket No. 062692-509001 WO

[0056] In some embodiments, the exon may include a nucleic acid sequence at least 75% identical to SEQ ID NO: 2. In some embodiments, the exon may include a nucleic acid sequence of at least 70% identical to SEQ ID NO: 2. A target nucleic acid sequence may target any of the aforementioned exon sequences. A target nucleic acid sequence may target a region of any of the aforementioned exon sequences.

[0057] SCN2A

[0058]

[0044] Some embodiments relate to sodium channel protein type 2 subunit alpha (SCN2A). For example, a method may be directed at reducing or preventing inclusion of a non-productive exon in a mature SCN2A mRNA, where inclusion of the non-productive exon results in nonsense-mediated decay (NMD). Some examples of SCN2A protein sequences are included at UniProt.org under accession numbers Q99250 (human) and B1AWN6 (mouse), as last updated as of the effective filing date. The SCN2A protein may include the amino acid sequence of SEQ ID NO: 11. The SCN2A protein may include an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. The SCN2A protein may include the amino acid sequence of SEQ ID NO: 12. The SCN2A protein may include an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

[0059]

[0045] SEQ ID NO: 11 (UniProt Q99250-1, human SCN2A protein):

[0060] MAQSVLVPPGPDSFRFFTRESLAAIEQRIAEEKAKRPKQERKDEDDENGPKPNSDLEAGKSLPFIYGDIP PEMVSVPLEDLDPYYINKKTFIVLNKGKAISRFSATPALYILTPFNPIRKLAIKILVHSLFNMLIMCTILTN CVFMTMSNPPDWTKNVEYTFTGIYTFESLIKILARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFVDLG NVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLRNKCLQW PPDNSSFEINITSFFNNSLDGNGTTFNRTVSIFNWDEYIEDKSHFYFLEGQNDALLCGNSSDAGQCPEGYI CVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLIL AVVAMAYEEQNQATLEEAEQKEAEFQQMLEQLKKQQEEAQAAAAAASAESRDFSGAGGIGVFSESSS VASKLSSKSEKELKNRRKKKKQKEQSGEEEKNDRVRKSESEDSIRRKGFRFSLEGSRLTYEKRFSSPHQ SLLSIRGSLFSPRRNSRASLFSFRGRAKDIGSENDFADDEHSTFEDNDSRRDSLFVPHRHGERRHSNVSQ ASRASRVLPILPMNGKMHSAVDCNGVVSLVGGPSTLTSAGQLLPEGTTTETEIRKRRSSSYHVSMDLLE DPTSRQRAMSIASILTNTMEELEESRQKCPPCWYKFANMCLIWDCCKPWLKVKHLVNLVVMDPFVDL AITICIVLNTLFMAMEHYPMTEQFSSVLSVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVSLS LMELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFG KSYKECVCKISNDCELPRWHMHDFFHSFLIVFRVLCGEWIETMWDCMEVAGQTMCLTVFMMVMVIG NLVVLNLFLALLLSSFSSDNLAATDDDNEMNNLQIAVGRMQKGIDFVKRKIREFIQKAFVRKQKALDEI KPLEDLNNKKDSCISNHTTIEIGKDLNYLKDGNGTTSGIGSSVEKYVVDESDYMSFINNPSLTVTVPIAV GESDFENLNTEEFSSESDMEESKEKLNATSSSEGSTVDIGAPAEGEQPEVEPEESLEPEACFTEDCVRKFK CCQISIEEGKGKLWWNLRKTCYKIVEHNWFETFIVFMILLSSGALAFEDIYIEQRKTIKTMLEYADKVFT Attorney Docket No. 062692-509001 WO

[0061] YIFILEMLLKWVAYGFQVYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSR

[0062] FEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINYTTGEMFDVSVVNNYSECKA

[0063] LIESNQTARWKNVKVNFDNVGLGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEDNLYMYLYFV

[0064] IFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPANKFQGMV

[0065] FDFVTKQVFDISIMILICLNMVTMMVETDDQSQEMTNILYWINLVFIVLFTGECVLKLISLRYYYFTIGW

[0066] NIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLL

[0067] FLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSGPPDCDPDKD

[0068] HPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDP

[0069] DATQFIEFAKLSDFADALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRI QMEERFMASNPSKVSYEPITTTLKRKQEEVSAIIIQRAYRRYLLKQKVKKVSSIYKKDKGKECDGTPIKE DTLIDKLNENSTPEKTDMTPSTTSPPSYDSVTKPEKEKFEKDKSEKEDKGKDIRESKK.

[0070]

[0046] SEQ ID NO: 12 (UniProt B1AWN6-1, mouse SCN2A protein):

[0071] MAQSVLVPPGPDSFRFFTRESLAAIEQRIAEEKAKRPKQERKDEDDENGPKPNSDLEAGKSLPFIYGDIP PEMVSEPLEDLDPYYINKKTFIVLNKGKAISRFSATSALYILTPFNPIRKLAIKILVHSLFNVLIMCTILTNC VFMTMSNPPDWTKNVEYTFTGIYTFESLIKILARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFVNLGN

[0072] VSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLRNKCLQWP

[0073] PDNSTFEINITSFFNNSLDWNGTAFNRTMNMFNWDEYIEDKSHFYFLEGQNDALLCGNSSDAGQCPEG YICVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLI LAVVAMAYEEQNQATLEEAEQKEAEFQQMLEQLKKQQEEAQAAAAAASAESRDFSGAGGIGVFSESS

[0074] SVASKLSSKSEKELKNRRKKKKQKEQAGEEEKEDAVRKSASEDSIRKKGFRFSLEGSRLTYEKRFSSPH QSLLSIRGSLFSPRRNSRASLFSFKGRVKDIGSENDFADDEHSTFEDNDSRRDSLFVPHRHGERRPSNVS QASRASRGIPTLPMNGKMHSAVDCNGVVSLVGGPSALTSPVGQLLPEGTTTETEIRKRRSSSYHVSMDL

[0075] LEDPTSRQRAMSMASILTNTMEELEESRQKCPPCWYKFANMCLIWDCCKPWLKVKHVVNLVVMDPF

[0076] VDLAITICIVLNTLFMAMEHYPMTEQFSSVLSVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFI

[0077] VSLSLMELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGM

[0078] QLFGKSYKECVCKISNDCELPRWHMHDFFHSFLIVFRVLCGEWIETMWDCMEVAGQTMCLTVFMMV

[0079] MVIGNLVVLNLFLALLLSSFSSDNLAATDDDNEMNNLQIAVGRMQKGIDFVKRKIREFIQKAFVRKQK

[0080] ALDEIKPLEDLNNKKDSCISNHTTIEIGKDLNYLKDGNGTTSGIGSSVEKYVVDESDYMSFINNPSLTVT

[0081] VPIAVGESDFENLNTEEFSSESDMEESKEKLNATSSSEGSTVDIGAPAEGEQPEAEPEESLEPEACFTEDC

[0082] VRKFKCCQISIEEGKGKLWWNLRKTCYKIVEHNWFETFIVFMILLSSGALAFEDIYIEQRKTIKTMLEYA

[0083] DKVFTYIFILEMLLKWVAYGFQMYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRP

[0084] LRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINYTTGEMFDVSVVNN

[0085] YSECQALIESNQTARWKNVKVNFDNVGLGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEDNLY

[0086] MYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPAN

[0087] KFQGMVFDFVTKQVFDISIMILICLNMVTMMVETDDQSQEMTNILYWINLVFIVLFTGECVLKLISLRH

[0088] YYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPA

[0089] LFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSGPPD

[0090] CDPEKDHPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEV

[0091] WEKFDPDATQFIEFCKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGE Attorney Docket No. 062692-509001 WO

[0092] MDALRIQMEERFMASNPSKVSYEPITTTLKRKQEEVSAIVIQRAYRRYLLKQKVKKVSSIYKKDKGKED EGTPIKEDIITDKLNENSTPEKTDVTPSTTSPPSYDSVTKPEKEKFEKDKSEKEDKGKDIRESKK.

[0093] Modified U7 snRNAs

[0094]

[0047] Disclosed herein, in some embodiments, are recombinant small nuclear RNAs (snRNAs) or snRNA sequences. Disclosed herein, in some embodiments, are modified or recombinant U7 snRNAs, modified or recombinant U7 snRNA sequences, and systems that include a modified or recombinant U7 snRNA or that include a modified or recombinant U7 snRNA sequence. The modified or recombinant U7 snRNA may be or include an engineered U7 snRNA. Terms such as modified, recombinant, and engineered may be used interchangeably herein. The U7 snRNA sequence may be useful for modifying nucleic acid splicing. In some embodiments, the U7 snRNA sequence includes an exonic splicing silencer (ESS) nucleic acid sequence. Some embodiments do not include an ESS nucleic acid sequence. In some embodiments, the U7 snRNA sequence may include a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA). In some embodiments, the target nucleic acid sequence may encode the sodium channel protein type 2 subunit alpha (SCN2A). The U7 snRNA sequence may include a smOPT sequence. The U7 snRNA sequence may include a hairpin.

[0095]

[0048] Described herein, in some embodiments, is a nucleic acid system that may include one or more RNA molecules. In some embodiments, the system may include at least one RNA molecule. In some embodiments, the system may include at least two RNA molecules. In some embodiments, the system may include at least three RNA molecules. In some embodiments, the system may include at least four RNA molecules. In some embodiments, the system may include at least five RNA molecules. In some embodiments, the system may include at most one RNA molecule. In some embodiments, the system may include at most two RNA molecules. In some embodiments, the system may include at most three RNA molecules. In some embodiments, the system may include at most four RNA molecules. In some embodiments, the system may include at most five RNA molecules.

[0096]

[0049] In some embodiments, the RNA may be a modified U7 snRNA. Some U rich small nuclear ribonucleoproteins (snRNPs) include complexes that mediate the splicing of pre-mRNAs. U7 snRNP may be an exception in not being involved in splicing but may be a key factor in 3’ end processing of replication-dependent histone mRNAs. However, by introducing controlled changes in the U7 snRNA histone binding sequence and in the Sm motif, U7 can be used as an effective tool for gene therapy. The modified U7 snRNP may thus be not involved in processing of replicationdependent histone pre-mRNA but instead target splicing by inducing efficient skipping or inclusion of selected exons. Modification of the sequence motif of U7 snRNA can make the snRNP particle hybridize to almost any RNA sequence within the nucleoplasm. Attorney Docket No. 062692-509001 WO

[0097]

[0050] A modified U7 snRNA may include, in the following order from 5’ to 3’: (1) an optional exonic splicing silencer, (2) a targeting sequence (e.g. an antisense sequence) complementary to a region of an SCN2A mRNA, (3) a smOPT sequence, and (4) a hairpin sequence.

[0098]

[0051] Some embodiments include a modified U7 small nuclear RNA (snRNA). In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 90% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 91% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 92% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 93% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 94% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 95% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 96% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 97% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 98% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence at least 99% identical to a U7 core sequence set forth in Table 1. In some embodiments, the modified U7 snRNA includes a U7 core sequence set forth in Table 1. A percent identity in relation to a U7 core sequence set forth in Table 1 may exclude the (N. . .N) region. In some embodiments, the modified U7 snRNA is a U7 core sequence set forth in Table 1. For the U7 core sequence shown in Table 1, an antisense sequence is in parentheses, a smOPT is in upper case, and a hairpin is in brackets. In the U7 Core Sequence in Table 1, “(N.. .N)” may denotes an insertion site of an ESS and U7 targeting sequence (e.g., set forth in Table 2-4), or an insertion site of a U7 targeting sequence.

[0099] Table 1. Sequences of functional elements useful for gene therapy

[0100]

[0052] Some embodiments include a modified U7 small nuclear RNA (snRNA). In some embodiments, the modified U7 snRNA includes a U7 sequence at least 90% identical to a U7 Attorney Docket No. 062692-509001 WO sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 91% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 92% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 93% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 94% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 95% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 96% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 97% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 98% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence at least 99% identical to a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA includes a U7 sequence set forth in Table 7B. In some embodiments, the modified U7 snRNA is a U7 sequence set forth in Table 7B.

[0101]

[0053] Some embodiments relate to a transcribable sequence. In some embodiments, the transcribable sequence encodes an inhibitory RNA. An example of an inhibitory RNA may include a modified snRNA. An example of an inhibitory RNA may include a modified U7 snRNA.

[0102]

[0054] Some embodiments relate to an inhibitory RNA such as a recombinant or modified snRNA (e.g., U7 snRNA). The inhibitory RNA may be encoded by a transcribable sequence. Disclosed herein, in some embodiments, are inhibitory RNAs. Disclosed herein, in some embodiments, are nucleic acids encoding an inhibitory RNA.

[0103] ESS Sequences

[0104]

[0055] Described herein, in some embodiments, are exonic splicing silencer (ESS) sequences. An ESS may be or include a 10-20 nt sequence at a 5’ terminus of an snRNA (e.g., engineered snRNA) capable of enhancing splicing suppression. The ESS may be included in a modified or recombinant U7 snRNA. Some embodiments omit an ESS. The ESS may recruit a protein factor that reduces splicing of the RNA encoding SCN2A. An ESS sequence may refer to an ESS or to a sequence that encodes an ESS. An ESS may include a short region of an exon and is a cis-regulatory element (CREs). CREs are regions of non-coding DNA which regulate transcription of neighboring genes. CREs may include components of genetic regulatory networks that control the timing and the amount that a specific gene is expressed. ESSs may work by inhibiting the splicing of pre-mRNA transcripts or promoting exon skipping. In some embodiments, the ESS may recruit a protein factor that silences splicing of a target RNA such as an RNA encoding SCN2A. In some embodiments, the ESS may recruit a group of factors that reduce splicing of the target RNA. In some embodiments, the ESS may recruit a group of factors that silence splicing of the RNA encoding SCN2A. Attorney Docket No. 062692-509001 WO

[0105]

[0056] In some embodiments, the ESS may be about 20 nucleotides long. In some embodiments, the ESS may be at least 4 nucleotides long. In some embodiments, the ESS may be at least 5 nucleotides long. In some embodiments, the ESS may be at least 6 nucleotides long. In some embodiments, the ESS may be at least 7 nucleotides long. In some embodiments, the ESS may be at least 8 nucleotides long. In some embodiments, the ESS may be at least 9 nucleotides long. In some embodiments, the ESS may be at least 10 nucleotides long. In some embodiments, the ESS may be at least 11 nucleotides long. In some embodiments, the ESS may be at least 12 nucleotides long. In some embodiments, the ESS may be at least 13 nucleotides long. In some embodiments, the ESS may be at least 14 nucleotides long. In some embodiments, the ESS may be at least 15 nucleotides long. In some embodiments, the ESS may be at least 16 nucleotides long. In some embodiments, the ESS may be at least 17 nucleotides long. In some embodiments, the ESS may be at least 18 nucleotides long. In some embodiments, the ESS may be at least 19 nucleotides long. In some embodiments, the ESS may be at least 20 nucleotides long.

[0106]

[0057] In some embodiments, the ESS nucleic acid sequence may include an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 99% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 98% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 97% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 96% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 95% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 94% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 93% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 92% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 91% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 90% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 85% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 80% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 75% identical to an ESS nucleic acid sequence in Table 2 or Table 4. In some embodiments, the ESS nucleic acid sequence Attorney Docket No. 062692-509001 WO may include a nucleic acid sequence at least 70% identical to an ESS nucleic acid sequence in Table 2 or Table 4.

[0107]

[0058] In some embodiments, the ESS nucleic acid sequence may be or include ATGATAGGGACTTAGGGTGA (SEQ ID NO: 13). In some embodiments, the ESS nucleic acid sequence may be or include TTTGTTCCGTGGGTGGTTTA (SEQ ID NO: 14). In some embodiments, the ESS nucleic acid sequence may be or include TGGGGGGAGGTAGGTAGGTA (SEQ ID NO: 15). In some embodiments, the ESS nucleic acid sequence may be or include TTTGTTCCGTTTCGTAGGTA (SEQ ID NO: 1136).

[0108]

[0059] In some embodiments, the ESS nucleic acid sequence may include the ESS nucleic acid sequence of SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 99% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 98% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 97% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 96% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 95% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 94% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 93% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 92% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 91% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 90% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 85% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 80% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 75% identical to SEQ ID NO: 1136. In some embodiments, the ESS nucleic acid sequence may include a nucleic acid sequence at least 70% identical to SEQ ID NO: 1136.

[0109]

[0060] In some embodiments, the inhibitory RNA comprises an exonic splicing silencer (ESS) sequence. The ESS sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to 5’-TTTGTTCCGTTTCGTAGGTA-3’ (SEQ ID NO: 1136), or to another ESS sequence herein. In some embodiments, the inhibitory RNA comprises an ESS sequence comprising the sequence of SEQ ID NO: 1136, or a sequence thereof having 1, 2, 3, 4, or 5 substitutions, deletions, or insertions. In some embodiments, the ESS sequence comprises a sequence having 1, 2, 3, or 4 substitutions, deletions, or insertions relative to SEQ ID NO: 1136. In some embodiments, the ESS Attorney Docket No. 062692-509001 WO sequence comprises a sequence having 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NO: 1136. In some embodiments, the ESS sequence comprises a sequence having 1 or 2 substitutions, deletions, or insertions relative to SEQ ID NO: 1136. In some embodiments, the ESS sequence comprises a sequence having 1 substitution, deletion, or insertion relative to SEQ ID NO: 1136.

[0110] U7 Targeting Sequences

[0111]

[0061] Described herein, in some embodiments, are targeting nucleic acid sequences such as snRNA targeting sequences or U7 targeting sequences. A targeting nucleic acid sequence may be or include an antisense nucleic acid sequence. A U7 targeting nucleic acid sequence may be or include a U7 antisense nucleic acid sequence. An snRNA targeting nucleic acid sequence may be or include a snRNA antisense nucleic acid sequence. Described herein, in some embodiments, are antisense nucleic acid sequences such as snRNA antisense sequences or U7 antisense sequences. An antisense sequence may be referred to as a targeting sequence. The antisense nucleic acid sequence may be included in a modified or recombinant snRNA or U7 snRNA. The targeting nucleic acid sequence may target (e.g., bind or be reverse complementary to) a target RNA such as an RNA encoding an SCN2A protein (Navi.2). The antisense nucleic acid sequence may bind or be reverse complementary to a target RNA such as an RNA encoding an SCN2A protein. The antisense nucleic acid sequence may bind to the target RNA. In some embodiments, an antisense nucleic acid sequence is encoded by a DNA sequence (e.g., a DNA expression construct). Targeting an RNA such as an SCN2A RNA may include binding or being reverse complementary to the target RNA.

[0112]

[0062] Described herein, in some embodiments, is a system wherein the target nucleic acid sequence may be 10-60 nucleotides in length. Some U rich small nuclear ribonucleoproteins (snRNPs) are complexes that may mediate the splicing of pre-mRNAs. U7 snRNP may be an exception in being uninvolved in splicing but may be a factor in unique 3’ end processing of replication-dependent histone mRNAs. However, by introducing controlled changes in the U7 snRNA histone binding sequence and in the Sm motif, U7 can be used as an effective tool for gene therapy. In some embodiments, the modified U7 snRNP may not be uninvolved in processing of replicationdependent histone pre-mRNA, but target splicing by inducing efficient skipping or inclusion of selected exons. Modification of the sequence motif of U7 snRNA can make the snRNP particle hybridize to almost any RNA sequence within the nucleoplasm. In some embodiments, the target nucleic sequence may be at least 10 nucleotides. In some embodiments, the target nucleic sequence may be at least 11 nucleotides. In some embodiments, the target nucleic sequence may be at least 12 nucleotides. In some embodiments, the target nucleic sequence may be at least 13 nucleotides. In some embodiments, the target nucleic sequence may be at least 14 nucleotides. In some embodiments, the target nucleic sequence may be at least 15 nucleotides. In some embodiments, the target nucleic sequence may be at least 16 nucleotides. In some embodiments, the target nucleic sequence may be at Attorney Docket No. 062692-509001 WO least 17 nucleotides. In some embodiments, the target nucleic sequence may be at least 18 nucleotides. In some embodiments, the target nucleic sequence may be at least 19 nucleotides. In some embodiments, the target nucleic sequence may be at least 20 nucleotides. In some embodiments, the target nucleic sequence may be at least 22 nucleotides. In some embodiments, the target nucleic sequence may be at least 24 nucleotides. In some embodiments, the target nucleic sequence may be at least 26 nucleotides. In some embodiments, the target nucleic sequence may be at least 28 nucleotides. In some embodiments, the target nucleic sequence may be at least 30 nucleotides. In some embodiments, the target nucleic sequence may be at least 32 nucleotides. In some embodiments, the target nucleic sequence may be at least 34 nucleotides. In some embodiments, the target nucleic sequence may be at least 36 nucleotides. In some embodiments, the target nucleic sequence may be at least 38 nucleotides. In some embodiments, the target nucleic sequence may be at least 40 nucleotides. In some embodiments, the target nucleic sequence may be at least 45 nucleotides. In some embodiments, the target nucleic sequence may be at least 50 nucleotides. In some embodiments, the target nucleic sequence may be at least 55 nucleotides. In some embodiments, the target nucleic sequence may be at least 60 nucleotides. In some embodiments, the target nucleic sequence may be at least 65 nucleotides. In some embodiments, the target nucleic sequence may be at least 70 nucleotides. In some embodiments, the target nucleic sequence may be at least 75 nucleotides. In some embodiments, the target nucleic sequence may be at least 80 nucleotides. In some embodiments, the target nucleic sequence may be at least 85 nucleotides. In some embodiments, the target nucleic sequence may be at least 90 nucleotides. In some embodiments, the target nucleic sequence may be at least 95 nucleotides. In some embodiments, the target nucleic sequence may be at least 100 nucleotides. In some embodiments, the target nucleic sequence may be at least 125 nucleotides. In some embodiments, the target nucleic sequence may be at least 150 nucleotides. In some embodiments, the target nucleic sequence may be at least 175 nucleotides. In some embodiments, the target nucleic sequence may be at least 200 nucleotides. In some embodiments, the target nucleic sequence may be at least 225 nucleotides. In some embodiments, the target nucleic sequence may be at least 250 nucleotides. In some embodiments, the target nucleic sequence may be at most 10 nucleotides. In some embodiments, the target nucleic sequence may be at most 11 nucleotides. In some embodiments, the target nucleic sequence may be at most 12 nucleotides. In some embodiments, the target nucleic sequence may be at most 13 nucleotides. In some embodiments, the target nucleic sequence may be at most 14 nucleotides. In some embodiments, the target nucleic sequence may be at most 15 nucleotides. In some embodiments, the target nucleic sequence may be at most 16 nucleotides. In some embodiments, the target nucleic sequence may be at most 17 nucleotides. In some embodiments, the target nucleic sequence may be at most 18 nucleotides. In some embodiments, the target nucleic sequence may be at most 19 nucleotides. In some embodiments, the target nucleic sequence may be at most 20 nucleotides. In some embodiments, the target nucleic sequence may be at most 22 nucleotides. In some embodiments, the target nucleic sequence may be at most 24 Attorney Docket No. 062692-509001 WO nucleotides. In some embodiments, the target nucleic sequence may be at most 26 nucleotides. In some embodiments, the target nucleic sequence may be at most 28 nucleotides. In some embodiments, the target nucleic sequence may be at most 30 nucleotides. In some embodiments, the target nucleic sequence may be at most 32 nucleotides. In some embodiments, the target nucleic sequence may be at most 34 nucleotides. In some embodiments, the target nucleic sequence may be at most 36 nucleotides. In some embodiments, the target nucleic sequence may be at most 38 nucleotides. In some embodiments, the target nucleic sequence may be at most 40 nucleotides. In some embodiments, the target nucleic sequence may be at most 45 nucleotides. In some embodiments, the target nucleic sequence may be at most 50 nucleotides. In some embodiments, the target nucleic sequence may be at most 55 nucleotides. In some embodiments, the target nucleic sequence may be at most 60 nucleotides. In some embodiments, the target nucleic sequence may be at most 65 nucleotides. In some embodiments, the target nucleic sequence may be at most 70 nucleotides. In some embodiments, the target nucleic sequence may be at most 75 nucleotides. In some embodiments, the target nucleic sequence may be at most 80 nucleotides. In some embodiments, the target nucleic sequence may be at most 85 nucleotides. In some embodiments, the target nucleic sequence may be at most 90 nucleotides. In some embodiments, the target nucleic sequence may be at most 95 nucleotides. In some embodiments, the target nucleic sequence may be at most 100 nucleotides. In some embodiments, the target nucleic sequence may be at most 125 nucleotides. In some embodiments, the target nucleic sequence may be at most 150 nucleotides. In some embodiments, the target nucleic sequence may be at most 175 nucleotides. In some embodiments, the target nucleic sequence may be at most 200 nucleotides. In some embodiments, the target nucleic sequence may be at most 225 nucleotides. In some embodiments, the target nucleic sequence may be at most 250 nucleotides. The target nucleic acid sequence may, in some embodiments, include a length defined by a range of any two of the aforementioned numbers of nucleotides.

[0113]

[0063] In some embodiments, the target nucleic acid sequence may bind to the alternatively spliced region. In some embodiments, the target nucleic acid sequence may bind to the alternatively spliced region at the 5’ end of the nucleic acid. In some embodiments, the target nucleic acid sequence may bind to the alternatively spliced region at the 3’ end of the nucleic acid. In some embodiments, the target nucleic acid sequence may bind to the alternatively spliced region in between the 5’ and 3’ ends of the nucleic acid.

[0114]

[0064] A target nucleic acid sequence may hybridize or bind to an alternatively spliced region. In some embodiments, the target nucleic acid sequence is fully reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid sequence is partially reverse complementary (e.g. at least 80% reverse complementary or at least 90% reverse complementary) to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 99% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 98% reverse complementary to a portion of the Attorney Docket No. 062692-509001 WO alternatively spliced region. In some embodiments, the target nucleic acid is at least 97% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 96% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 95% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 94% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 93% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 92% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 91% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 90% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 85% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 80% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 75% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid is at least 70% reverse complementary to a portion of the alternatively spliced region. In some embodiments, the target nucleic acid sequence is fully reverse complementary or partially reverse complementary (e.g. at least 90% reverse complementary) to a portion of the alternatively spliced region.

[0115]

[0065] In some embodiments, the portion of the alternatively spliced region is within a 5’ half of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region is within a 5’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region is within a 5’ half or 5’ end of the alternatively spliced region.

[0116]

[0066] In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 50 base pairs (bp) of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 45 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 40 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 35 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 30 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 25 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 20 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region comprises nucleotide positions within 15 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of Attorney Docket No. 062692-509001 WO the alternatively spliced region comprises nucleotide positions within 10 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 45 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 40 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 35 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 30 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 25 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 20 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 15 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the portion of the alternatively spliced region excludes nucleotide positions within 10 bp of a 5’ or 3’ end of the alternatively spliced region. In some embodiments, the 5’ or 3’ end is a 5’ end. In some embodiments, the 5’ or 3’ end is a 3’ end.

[0117]

[0067] In some embodiments, the target nucleic acid sequence targets a targeted region of SCN2A. In some embodiments, the targeted region is within an exon of the endogenous SCN2A RNA. In some embodiments, the targeted region is within an exon of an endogenous SCN2A mRNA. In some embodiments, the target nucleic acid sequence targets an alternatively spliced exon of the endogenous SCN2A RNA. In some embodiments, the alternatively spliced exon includes an exon 13N of SCN2A.

[0118]

[0068] In some embodiments, the targeted region is within a 5’ half or 5’ end of an intron or exon of the endogenous SCN2A RNA. For example, in some embodiments, the targeted region may be closer to the 5’ end of an intron of the endogenous SCN2A RNA. In some embodiments, the targeted region includes the 5’ end of an intron of the endogenous SCN2A RNA. In some embodiments, the targeted region may be closer to the 5’ end of an exon of the endogenous SCN2A RNA. In some embodiments, the targeted region includes the 5’ end of an exon of the endogenous SCN2A RNA.

[0119]

[0069] In some embodiments, the targeted region is within a 3’ half or 3’ end of an intron or exon of the endogenous SCN2A RNA. For example, in some embodiments, the targeted region may be closer to the 3’ end of an intron of the endogenous SCN2A RNA. In some embodiments, the targeted region includes the 3’ end of an intron of the endogenous SCN2A RNA. In some embodiments, the targeted region may be closer to the 3’ end of an exon of the endogenous SCN2A RNA. In some embodiments, the targeted region includes the 3’ end of an exon of the endogenous SCN2A RNA. Attorney Docket No. 062692-509001 WO

[0120]

[0070] In some embodiments, the targeted region is within 100 nucleotides of an intron / exon junction. In some embodiments, the targeted region is within 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of an intron / exon junction. In some embodiments, the targeted region is not within 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of an intron / exon junction.

[0121]

[0071] In some embodiments, the targeted region of the endogenous SCN2A RNA (e.g., SCN2A RNA) includes an intron-exon junction of the endogenous RNA. The intron-exon junction refers to the boundary between an intron and exon and includes the splice site that separates the intron and the exon upon pre-mRNA splicing. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 10 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 20 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 30 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 40 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 40 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 60 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 70 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 80 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 90 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 100 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 110 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 120 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 130 nt and 150 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 140 nt and 150 nt of an intron-exon junction.

[0122]

[0072] In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 140 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 130 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 120 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 110 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 100 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 90 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 80 nt of an intron-exon Attorney Docket No. 062692-509001 WO junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 70 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 60 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 50 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 40 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 30 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 20 nt of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt and 10 nt of an intron-exon junction.

[0123]

[0073] In some embodiments, the targeted region of the endogenous SCN2A RNA is within 0 nt, 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, or 150 of an intron-exon junction. In some embodiments, the targeted region of the endogenous SCN2A RNA includes an intro-exon junction.

[0124]

[0074] In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence 100% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 99% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 98% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 97% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 96% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 95% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 94% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 93% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 92% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 91% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 90% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 85% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 80% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 75% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid Attorney Docket No. 062692-509001 WO sequence may include a nucleic acid sequence at least 70% identical to a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid targets or binds with an RNA that includes reverse complement of any of the aforementioned sequences.

[0125]

[0075] In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence 100% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 99% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 98% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 97% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 96% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 95% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 94% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 93% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 92% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 91% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 90% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 85% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 80% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 75% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 70% identical to a human version of a target nucleic acid sequence in Table 2. In some embodiments, the target nucleic acid targets or binds with an RNA that includes reverse complement of any of the aforementioned sequences.

[0126]

[0076] In some embodiments, the target nucleic acid sequence is associated with a relative normalized luciferase value in Table 2 that is at least 1.10, at least 1.20, at least 1.30, at least 1.40, at least 1.50, at least 1.60, or at least 1.70. In some embodiments, the target nucleic acid sequence is associated with a relative normalized luciferase value in Table 2 that is above 1.10, above 1.20, above 1.30, above 1.40, above 1.50, above 1.60, or above 1.70. The target nucleic acid sequence may be Attorney Docket No. 062692-509001 WO associated with a relative normalized luciferase value in Table 2 that is at least 1.50. The target nucleic acid sequence may be associated with a relative normalized luciferase value in Table 2 that is above 1.50.

[0127]

[0077] In some embodiments, the target nucleic acid sequence may include the nucleic acid sequence of a sequence in Table 3, or a reverse complement thereof. In some embodiments, the target nucleic acid sequence is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence in Table 3 or a reverse complement thereof, or a range of % identities defined by any two of the aforementioned percentages. In some embodiments, the target nucleic acid sequence may include the nucleic acid sequence of a human version of a sequence in Table 3, or a reverse complement thereof. In some embodiments, the target nucleic acid sequence is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a human version of a sequence in Table 3 or a reverse complement thereof, or a range of % identities defined by any two of the aforementioned percentages. In some embodiments, the target nucleic acid targets or binds with an RNA that includes reverse complement of any of the aforementioned sequences.

[0128]

[0078] In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence 100% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 99% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 98% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 97% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 96% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 95% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 94% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 93% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 92% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 91% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 90% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 85% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 80% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 75% Attorney Docket No. 062692-509001 WO identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid sequence may include a nucleic acid sequence at least 70% identical to a target nucleic acid sequence in Table 4. In some embodiments, the target nucleic acid targets or binds with an RNA that includes reverse complement of any of the aforementioned sequences.

[0129]

[0079] In some embodiments, the target nucleic acid sequence is associated with a relative normalized luciferase value in Table 4 that is at least 1.10, at least 1.20, at least 1.30, at least 1.40, at least 1.50, at least 1.60, or at least 1.70. In some embodiments, the target nucleic acid sequence is associated with a relative normalized luciferase value in Table 4 that is above 1.10, above 1.20, above 1.30, above 1.40, above 1.50, or above 1.60. The target nucleic acid sequence may be associated with a relative normalized luciferase value in Table 4 that is at least 1.50. The target nucleic acid sequence may be associated with a relative normalized luciferase value in Table 4 that is above 1.50.

[0130]

[0080] In some embodiments, the target nucleic acid sequence may include the nucleic acid sequence of a sequence in Table 15A, or a reverse complement thereof. In some embodiments, the target nucleic acid sequence is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence in Table 15A or a reverse complement thereof, or a range of % identities defined by any two of the aforementioned percentages. In some embodiments, the target nucleic acid sequence may include the nucleic acid sequence of a human version of a sequence in Table 15A, or a reverse complement thereof. In some embodiments, the target nucleic acid sequence is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a human version of a sequence in Table 15A or a reverse complement thereof, or a range of % identities defined by any two of the aforementioned percentages. In some embodiments, the target nucleic acid targets or binds with an RNA that includes reverse complement of any of the aforementioned sequences.

[0131]

[0081] The targeting nucleic acid sequence may bind to or be reverse complementary to a targeted region. In some embodiments, the targeting nucleic acid sequence binds to an SCN2A mRNA at a region 5’ or 3’ relative to exon 13N. The targeting nucleic acid sequence may bind to an SCN2A mRNA comprising the sequence of SEQ ID NO: 766. The targeting nucleic acid sequence may bind to an SCN2A mRNA comprising the sequence of SEQ ID NO: 767.

[0132]

[0082] In some embodiments, the targeting nucleic acid sequence binds to an SCN2A pre- mRNA at a region 5’ relative to exon 13N. The region may be up to 25 nucleotides, up to 50 nucleotides, up to 75 nucleotides, up to 100 nucleotides, or more, upstream from or 5’ to exon 13N in an SCN2A pre-mRNA.

[0133]

[0083] In some embodiments, the targeting nucleic acid sequence binds to an SCN2A pre- mRNA at a region 3’ relative to exon 13N. The region may be up to 25 nucleotides, up to 50 nucleotides, up to 75 nucleotides, up to 100 nucleotides, or more, downstream from or 3’ to exon 13N in an SCN2A pre-mRNA. Attorney Docket No. 062692-509001 WO

[0134]

[0084] In some embodiments, the engineered U7 snRNA includes a targeting nucleic acid sequence 3’ to the ESS nucleic acid sequence. In some embodiments, the engineered U7 snRNA includes a targeting nucleic acid sequence downstream relative to the ESS nucleic acid sequence. In some embodiments, the engineered U7 snRNA includes a targeting nucleic acid sequence 3’ or downstream relative to the ESS nucleic acid sequence.

[0135] Sm Binding Sites

[0136]

[0085] Described herein, in some embodiments, are systems that include a Sm binding site. Sm proteins may bind to a U7 snRNA via Sm binding site. Once the Sm proteins bind the U7 snRNA in the cytoplasm they may bind to a pre-mRNA and regulate splicing. In some embodiments, the system contains a Sm-like binding site. In some embodiments, the Sm binding site comprises AAUUUGUCUAG (SEQ ID NO: 112). In some embodiments, the Sm binding site comprises AAUUUUUGGAG (SEQ ID NO: 113; smOPT). A modified or recombinant U7 snRNA sequence may include a Sm binding site. A system may encode a modified or recombinant U7 snRNA sequence that includes a Sm binding site.

[0137]

[0086] In some embodiments, the Sm binding site may be 3’ relative to the ESS nucleic acid sequence. In some embodiments, the Sm binding site may be downstream relative to the ESS nucleic acid sequence. In some embodiments, the Sm binding site may be 3’ relative to the target nucleic acid sequence. In some embodiments, the Sm binding site may be downstream relative to the target nucleic acid sequence. In some embodiments, the Sm binding site is 3’ or downstream relative to the ESS nucleic acid sequence or target nucleic acid sequence.

[0138] U73 ’ Hairpins

[0139]

[0087] Described herein, in some embodiments, are systems that include a hairpin sequence. The hairpin sequence may include a U7 hairpin sequence. The U7 hairpin sequence may be a 3’ U7 hairpin sequence. A modified or recombinant U7 snRNA sequence may include the hairpin sequence. A system may encode a modified or recombinant U7 snRNA sequence that includes a hairpin sequence.

[0140]

[0088] Described herein, in some embodiments, is a nucleic acid system that may contain a hairpin sequence comprising a U7 small nuclear RNA (snRNA) hairpin sequence. A hairpin may include an unpaired loop of RNA that is created when a RNA strand folds and forms complementary base pairs with another section of the same strand. The resulting structure may look like a loop or a U- shape. Hairpins may include a common type of secondary structure in RNA molecules and can be formed when two complementary sequences in a single RNA molecule meet and bind each other.

[0141]

[0089] In some embodiments, the U7 snRNA hairpin sequence comprises the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the U7 snRNA hairpin sequence comprises a Attorney Docket No. 062692-509001 WO nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

[0142]

[0090] In some embodiments, the hairpin sequence may include a 3’ hairpin sequence. In some embodiments, the hairpin sequence may be 3’ or downstream relative to the ESS nucleic acid sequence. In some embodiments, the hairpin sequence may be 3’ or downstream relative to the target nucleic acid sequence. In some embodiments, the hairpin sequence may be 3’ or downstream relative to or the Sm binding site. In some embodiments, the hairpin sequence is 3’ or downstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, or the Sm binding site.

[0143] Expression Constructs

[0144]

[0091] Described herein, in some embodiments, are expression constructs. An expression construct may include an expression cassette. Some embodiments relate to a nucleic acid expression system. The expression construct may be DNA. The expression construct may encode an RNA system described herein. The expression construct may encode an RNA such as a modified U7 snRNA.

[0145]

[0092] In some embodiments, the system may include an expression cassette. In some embodiments, the expression cassette may include a promoter. In some embodiments, the expression cassette may include an exonic splicing sequence. In some embodiments, the expression cassette may include a U7 target sequence. In some embodiments, the expression cassette may include a smOPT. In some embodiments, the expression cassette may include a U7 3’ hairpin structure. In some embodiments, the expression cassette may include a 3’ terminator sequence. In some embodiments, the expression cassette may include a combination of two or more of the following: a promoter, an exonic splicing sequence, a U7 target sequence, a smOPT, a U7 3’ hairpin structure, and a 3’ terminator sequence.

[0146]

[0093] Some embodiments include an arrayed series of modified U7 snRNAs. In some embodiments, the array includes multiple U7 modules. Each module may include a target sequence. The target sequences of multiple modules may be the same. The target sequences of some modules may be different.

[0147]

[0094] A modified U7 snRNA may be expressed from a U7 cassette. A functional U7 cassette may include, in the following order from 5’ to 3’: (1) a promoter, (2) an optional exonic splicing silencer, (3) a targeting sequence complementary to a region of a target mRNA (e.g., SCN2A mRNA), (4) an smOPT sequence, (5) a hairpin sequence, and (6) a 3’ termination signal. The expressed functional U7 snRNA consists of, in the following order from 5’ to 3’: (1) an optional exonic splicing silencer, (2) a targeting sequence complementary to a region of target mRNA (e.g., SCN2A mRNA), (3) an smOPT sequence, and (4) a hairpin sequence.

[0148]

[0095] Disclosed herein, in some embodiments, are nucleic acid expression systems, comprising: a promoter and terminator operably linked to a transcribable sequence encoding an inhibitory RNA that increases SCN2A expression, the promoter and terminator being selected from (i)-(iv): a first Attorney Docket No. 062692-509001 WO promoter comprising a U12 small nuclear RNA (RNU12) promoter sequence, and a first terminator comprising a U1 small nuclear RNA 2 (RNU1-2) terminator sequence; a second promoter comprising a U3B small nuclear RNA 1 (RNU3bl) promoter sequence, and a second terminator comprising a U5A small nuclear 1 RNA (RNU5A-1) terminator sequence; a third promoter comprising a U2 small nuclear RNA 10 (RNU2-10) promoter sequence, and a third terminator comprising a U5g small nuclear RNA (RNU5g) terminator sequence; or a fourth promoter comprising a RNU2-10 promoter sequence, and a fourth terminator comprising a RNU12 terminator sequence.

[0149]

[0096] In some embodiments, the first promoter and the first terminator are from different organism species. In some embodiments, the second promoter and the second terminator are from different organism species. In some embodiments, the third promoter and the third terminator are from different organism species. In some embodiments, the fourth promoter and the fourth terminator are from different organism species.

[0150] Promoters

[0151]

[0097] Described herein, in some embodiments, is a nucleic acid system that may contain a promoter sequence. The promoter may be a part of an expression construct. A promoter may include a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or snRNA. Promoters may be located near the transcription start sites of genes.

[0152]

[0098] In some embodiments, the promoter is directly upstream of the open reading frame (ORF). In some embodiments, the promoter is 5 bps upstream of the ORF. In some embodiments, the promoter is 10 bps upstream of the ORF. In some embodiments, the promoter is 15 bps upstream of the ORF. In some embodiments, the promoter is 20 bps upstream of the ORF. In some embodiments, the promoter is 25 bps upstream of the ORF. In some embodiments, the promoter is 30 bps upstream of the ORF. In some embodiments, the promoter is 35 bps upstream of the ORF. In some embodiments, the promoter is 40 bps upstream of the ORF. In some embodiments, the promoter is 45 bps upstream of the ORF. In some embodiments, the promoter is 50 bps upstream of the ORF. In some embodiments, the promoter is 55 bps upstream of the ORF. In some embodiments, the promoter is 60 bps upstream of the ORF. In some embodiments, the promoter is 65 bps upstream of the ORF. In some embodiments, the promoter is 70 bps upstream of the ORF. In some embodiments, the promoter is 75 bps upstream of the ORF. In some embodiments, the promoter is 80 bps upstream of the ORF. In some embodiments, the promoter is 85 bps upstream of the ORF. In some embodiments, the promoter is 90 bps upstream of the ORF. In some embodiments, the promoter is 95 bps upstream of the ORF. In some embodiments, the promoter is 100 bps upstream of the ORF. In some embodiments, the promoter is 200 bps upstream of the ORF. In some embodiments, the promoter is 300 bps upstream of the ORF. In some embodiments, the promoter is 400 bps upstream of the ORF. In some embodiments, Attorney Docket No. 062692-509001 WO the promoter is 500 bps upstream of the ORF. In some embodiments, the promoter is 600 bps upstream of the ORF. In some embodiments, the promoter is 700 bps upstream of the ORF. In some embodiments, the promoter is 800 bps upstream of the ORF. In some embodiments, the promoter is 900 bps upstream of the ORF. In some embodiments, the promoter is 1000 bps upstream of the ORF. Promoters can be about 100-1000 base pairs long, the sequence of which is highly dependent on the gene and product of transcription, type or class of RNA polymerase recruited to the site, and species of organism. In some embodiments, the promoter is 100 base pairs long. In some embodiments, the promoter is 200 base pairs long. In some embodiments, the promoter is 300 base pairs long. In some embodiments, the promoter is 400 base pairs long. In some embodiments, the promoter is 500 base pairs long. In some embodiments, the promoter is 600 base pairs long. In some embodiments, the promoter is 700 base pairs long. In some embodiments, the promoter is 800 base pairs long. In some embodiments, the promoter is 900 base pairs long. In some embodiments, the promoter is 1000 base pairs long. In some embodiments, the promoter sequence may include a mouse or human promoter sequence.

[0153]

[0099] In some embodiments, the promoter sequence may include a mouse promoter sequence. In some embodiments, the promoter sequence may include a human promoter sequence. In some embodiments, the promoter sequence may include an snRNA promoter sequence. In some embodiments, the promoter sequence may include a U7 snRNA promoter sequence. In some embodiments, the promoter sequence may include a U1 promoter sequence. In some embodiments, the promoter sequence may include a mouse U7 snRNA (“Mm U7”) promoter sequence. In some embodiments, the promoter sequence may include a human U7 snRNA (“Hs U7”) promoter sequence. In some embodiments, the promoter sequence may include a mouse Ulal (“mulal” or “Mm Ulal”) promoter sequence. In some embodiments, the promoter sequence may include a human Ul-1 (“HUI” or “Hs Ul-1”) promoter sequence. In some embodiments, the promoter sequence may include a fragment or combination of fragments of the promoters listed above. In some embodiments, the promoter sequence comprises a mouse U7 snRNA (“Mm U7”) promoter sequence, a human U7 snRNA (“Hs U7”) promoter sequence, a mouse Ulal (“mulal” or “Mm Ulal”) promoter sequence, or a human Ul-1 (“HUI” or “Hs Ul-1”) promoter sequence, or a fragment or combination of fragments thereof. For the modified Mm U7 promoter of SEQ ID NO: 8, a U7 distal sequence element has been replaced with that of human Ul-1 and a U7 proximal sequence element has been replaced with that of mouse Ulal.

[0154]

[0100] In some embodiments, the promoter sequence may include a U7 snRNA promoter sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 promoter sequence. In some embodiments, the promoter sequence may include a U7 snRNA promoter sequence having a DSE replaced with a Ulal promoter sequence. In some embodiments, the promoter sequence comprises a U7 snRNA promoter sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 or Ulal promoter sequence. The promoter DSE is a region of the DNA that is Attorney Docket No. 062692-509001 WO normally found upstream of the open reading frame (ORF). The DSE is usually found upstream, from -250 to -170 bp. In some embodiments, the DSE is at least 10 base pairs long. In some embodiments, the DSE is at least 20 base pairs long. In some embodiments, the DSE is at least 30 base pairs long. In some embodiments, the DSE is at least 40 base pairs long. In some embodiments, the DSE is at least 50 base pairs long. In some embodiments, the DSE is at least 60 base pairs long. In some embodiments, the DSE is at least 70 base pairs long. In some embodiments, the DSE is at least 80 base pairs long. In some embodiments, the DSE is at least 90 base pairs long. In some embodiments, the DSE is at least 100 base pairs long. In some embodiments, the DSE is at least 110 base pairs long. In some embodiments, the DSE is at least 120 base pairs long. In some embodiments, the DSE is at least 130 base pairs long. In some embodiments, the DSE is at least 140 base pairs long. In some embodiments, the DSE is at least 150 base pairs long. In some embodiments, the DSE is at least 160 base pairs long.

[0155]

[0101] In some embodiments, the promoter sequence may include a mouse U7 promoter sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 promoter sequence. In some embodiments, the promoter sequence may include a mouse U7 promoter sequence having a PSE replaced with a Ulal promoter sequence. In some embodiments, the promoter sequence comprises a mouse U7 promoter sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 or Ulal promoter sequence. The promoter PSE may be an essential promoter element located 40-70 bp upstream of the ORF. The promoter PSE may be recognized by a specific transcription factor, snRNA activating protein complex (SNAPc). In some embodiments, the promoter PSE is 5 base pairs long. In some embodiments, the promoter PSE is 10 base pairs long. In some embodiments, the promoter PSE is 15 base pairs long. In some embodiments, the promoter PSE is 20 base pairs long. In some embodiments, the promoter PSE is 25 base pairs long. In some embodiments, the promoter PSE is 30 base pairs long. In some embodiments, the promoter PSE is 35 base pairs long. In some embodiments, the promoter PSE is 40 base pairs long. In some embodiments, the promoter PSE is 45 base pairs long. In some embodiments, the promoter PSE is 50 base pairs long. In some embodiments, the promoter PSE is 55 base pairs long. In some embodiments, the promoter PSE is 60 base pairs long.

[0156]

[0102] In some embodiments, the promoter sequence may include a nucleic acid sequence identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 99% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 98% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 97% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 96% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 95% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may Attorney Docket No. 062692-509001 WO include a nucleic acid sequence at least 94% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 93% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 92% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 91% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 90% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 85% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 80% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 75% identical to a promoter sequence in Table 5. In some embodiments, the promoter sequence may include a nucleic acid sequence at least 70% identical to a promoter sequence in Table 5. For the modified Mm U7 promoter in Table 5 (SEQ ID NO: 8), a U7 distal sequence element has been replaced with that of human Ul-1 and a U7 proximal sequence element has been replaced with that of mouse Ulal.

[0157]

[0103] A promoter may be modified. A promoter may be engineered. Some examples of modified or engineered promoters are shown in Table 14B. Such promoters may be included in an expression construct, or to drive expression of a transcript (such as an engineered U7 snRNA). In some embodiments, the promoter sequence may include a nucleic acid sequence identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 99% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 98% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 97% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 96% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 95% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 94% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 93% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 92% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 91% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 90% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 85% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 80% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least Attorney Docket No. 062692-509001 WO

[0158] 75% identical to a promoter sequence in Table 14B. In some embodiments, the promoter sequence includes a nucleic acid sequence at least 70% identical to a promoter sequence in Table 14B.

[0159]

[0104] Some examples of promoters include an RNU12 promoter, an RNU3bl promoter, or an RNU2-10 promoter. For example, a promoter may be or include an hRNU12 promoter, an mRNU3bl promoter, or an mRNU2-10 promoter. In some embodiments, a promoter includes an mRNU12 promoter, an hRNU3bl promoter, or an hRNU2-10 promoter. The promoter may include a functional fragment thereof.

[0160]

[0105] In some embodiments, a system includes a truncated small nuclear RNA (snRNA) promoter. The promoter may be operably linked to a transcribable sequence. The truncated snRNA promoter may be or include a functional fragment of a promoter sequence. The truncated snRNA promoter may be or include a functional fragment of an RNU12 promoter sequence. The truncated snRNA promoter may be or include a functional fragment of a RNU3bl promoter sequence. The truncated snRNA promoter may be or include a functional fragment of a RNU2-10 promoter sequence.

[0161]

[0106] A promoter may be or include an RNU12 promoter. The promoter may be or include an hRNU12 promoter. The promoter may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an hRNU12 promoter sequence provided herein. The promoter may be or include a functional fragment of a promoter. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated hRNU12 promoter sequence provided herein.

[0162]

[0107] A promoter may be or include an RNU3bl promoter. The promoter may be or include an mRNU3bl promoter. The promoter may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an mRNU3bl promoter sequence provided herein. The promoter may be or include a functional fragment of a promoter. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated mRNU3bl promoter sequence provided herein.

[0163]

[0108] A promoter may be or include an RNU2-10 promoter. The promoter may be or include an mRNU2-10 promoter. The promoter may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an mRNU2-10 promoter sequence provided herein. The promoter may be or include a functional fragment of a promoter. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated mRNU2-10 promoter sequence provided herein.

[0164]

[0109] In some embodiments, the RNU12 promoter sequence comprises a human RNU12 promoter (hRNU12) sequence. In some embodiments, the hRNU12 promoter sequence is at least Attorney Docket No. 062692-509001 WO

[0165] 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an hRNU12 promoter sequence herein, or a functional fragment thereof. In some embodiments, the RNU3bl promoter sequence comprises a mouse RNU3bl promoter (mRNU3bl) sequence. In some embodiments, the mRNU3bl promoter sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an mRNU3bl promoter sequence herein, or a functional fragment thereof. In some embodiments, the RNU2-10 promoter sequence comprises a mouse RNU2- 10 promoter (mRNU2-10) sequence. In some embodiments, the mRNU2-10 promoter sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a mRNU2-10 promoter sequence herein, or a functional fragment thereof.

[0166] [HO] A recombinant promoter may be or include a functional fragment. In some embodiments, a promoter is or includes a functional promoter fragment. In some embodiments, the functional fragment is about 50 base pairs (bp), about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp, or a range defined by any 2 of the aforementioned bp lengths. In some embodiments, the functional fragment is less than 75 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, or less than 500 bp. In some embodiments, the functional fragment is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, or at least 450 bp. In some embodiments, the functional fragment is a 5’ fragment. In some embodiments, the functional fragment is a 3’ fragment. In some embodiments, the functional fragment is an internal fragment.

[0167]

[0111] Some embodiments include 1 promoter. Some embodiments include at least 1 promoter. Some embodiments have no more than 1 promoter. Some embodiments include 2 promoters. Some embodiments include at least 2 promoters. Some embodiments have no more than 2 promoters. Some embodiments include 3 promoters. Some embodiments include at least 3 promoters. Some embodiments have no more than 3 promoters. Some embodiments include 4 promoters. Some embodiments include at least 4 promoters. Some embodiments have no more than 4 promoters. Some embodiments include 5 promoters. Some embodiments include at least 5 promoters. Some embodiments have no more than 5 promoters.

[0168]

[0112] In some embodiments, the promoter sequence may be 5’ or upstream relative to the ESS nucleic acid sequence. In some embodiments, the promoter sequence may be 5’ or upstream relative to the target nucleic acid sequence. In some embodiments, the promoter sequence may be 5’ or upstream relative to the Sm binding site. In some embodiments, the promoter sequence may be 5’ or upstream relative to the hairpin sequence. In some embodiments, the promoter sequence is 5’ or Attorney Docket No. 062692-509001 WO upstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, the Sm binding site, or the hairpin sequence.

[0169] 3 ’ Terminator Sequences

[0170]

[0113] Described herein, in some embodiments, is a nucleic acid system that may contain a terminator sequence. The terminator sequence may be a part of an expression construct. Described herein, in some embodiments, the nucleic acid system may include a terminator sequence. The terminator sequence may include a region of a nucleic acid sequence that marks the end of a gene or operon during transcription. The terminator sequence may mediate transcriptional termination by providing signals in the newly synthesized transcript RNA that triggers processes which release the transcript RNA from the transcriptional complex.

[0171]

[0114] In some embodiments, the terminator sequence may include a mouse or human terminator sequence. In some embodiments, the terminator sequence may include an snRNA terminator sequence. In some embodiments, the terminator sequence may include a U7 snRNA terminator sequence. In some embodiments, the terminator sequence may include a U1 terminator sequence. In some embodiments, the terminator sequence may include a Mm U7 terminator sequence. In some embodiments, the terminator sequence may include a Hs U7 terminator sequence. In some embodiments, the terminator sequence may include a mulal terminator sequence. In some embodiments, the terminator sequence may include a HUI terminator sequence. In some embodiments, the terminator sequence may include a fragment or a combination of fragments of the terminators listed above. In some embodiments, the terminator sequence comprises a Mm U7 terminator sequence, a Hs U7 terminator sequence, a mulal terminator sequence, or a HUI terminator sequence, or a fragment or combination of fragments thereof.

[0172]

[0115] In some embodiments, the terminator sequence comprises a U7 snRNA terminator sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 terminator sequence. In some embodiments, the terminator sequence comprises a U7 snRNA terminator sequence having a DSE replaced with a DSE of a Ulal terminator sequence. In some embodiments, the terminator sequence comprises a U7 snRNA terminator sequence having a distal sequence element (DSE) replaced with a DSE of a Ul-1 or Ulal terminator sequence. The terminator DSE is a region of the DNA that is normally found downstream of the snRNA at the position from +250 to +170 bp. In some embodiments, the terminator DSE is at least 10 base pairs long. In some embodiments, the terminator DSE is at least 20 base pairs long. In some embodiments, the terminator DSE is at least 30 base pairs long. In some embodiments, the terminator DSE is at least 40 base pairs long. In some embodiments, the terminator DSE is at least 50 base pairs long. In some embodiments, the terminator DSE is at least 60 base pairs long. In some embodiments, the terminator DSE is at least 70 base pairs long. In some embodiments, the terminator DSE is at least 80 base pairs long. In some embodiments, the terminator DSE is at least 90 base pairs long. In some embodiments, the terminator DSE is at least Attorney Docket No. 062692-509001 WO

[0173] 100 base pairs long. In some embodiments, the terminator DSE is at least 110 base pairs long. In some embodiments, the terminator DSE is at least 120 base pairs long. In some embodiments, the terminator DSE is at least 130 base pairs long. In some embodiments, the terminator DSE is at least 140 base pairs long. In some embodiments, the terminator DSE is at least 150 base pairs long. In some embodiments, the terminator DSE is at least 160 base pairs long.

[0174]

[0116] In some embodiments, the terminator sequence may include a mouse U7 snRNA terminator sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 terminator sequence. In some embodiments, the terminator sequence may include a mouse U7 snRNA terminator sequence having a PSE replaced with a PSE of a Ulal terminator sequence. In some embodiments, the terminator sequence comprises a mouse U7 snRNA terminator sequence having a proximal sequence element (PSE) replaced with a PSE of a Ul-1 or Ulal terminator sequence. The terminator PSE may be an essential terminator element located 40-70 bp downstream of the terminator start site. In some embodiments, the terminator PSE is 5 base pairs long. In some embodiments, the terminator PSE is 10 base pairs long. In some embodiments, the terminator PSE is 15 base pairs long. In some embodiments, the terminator PSE is 20 base pairs long. In some embodiments, the terminator PSE is 25 base pairs long. In some embodiments, the terminator PSE is 30 base pairs long. In some embodiments, the terminator PSE is 35 base pairs long. In some embodiments, the terminator PSE is 40 base pairs long. In some embodiments, the terminator PSE is 45 base pairs long. In some embodiments, the terminator PSE is 50 base pairs long. In some embodiments, the terminator PSE is 55 base pairs long. In some embodiments, the terminator PSE is 60 base pairs long.

[0175]

[0117] In some embodiments, the terminator sequence may include a nucleic acid sequence. In some embodiments, the terminator sequence may include a nucleic acid sequence identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 99% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 98% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 97% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 96% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 95% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 94% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 93% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 92% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 91% identical to a terminator sequence in Table 5. In some embodiments, the Attorney Docket No. 062692-509001 WO terminator sequence may include a nucleic acid sequence at least 90% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 85% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 80% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 75% identical to a terminator sequence in Table 5. In some embodiments, the terminator sequence may include a nucleic acid sequence at least 70% identical to a terminator sequence in Table 5.

[0176]

[0118] A terminator may be modified. A terminator may be engineered. Some examples of modified or engineered terminators are shown in Table 14C. Such terminators may be included in an expression construct, or to affect expression of a transcript (such as an engineered U7 snRNA). In some embodiments, the terminator sequence may include a nucleic acid sequence identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 99% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 98% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 97% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 96% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 95% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 94% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 93% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 92% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 91% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 90% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 85% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 80% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 75% identical to a terminator sequence in Table 14C. In some embodiments, the terminator sequence includes a nucleic acid sequence at least 70% identical to a terminator sequence in Table 14C.

[0177]

[0119] Some examples of terminators include an RNU1-2 terminator, an RNU5A-1 terminator, an RNU5g terminator, or an RNU12 terminator. For example, a terminator may be or include an hRNUl-2 terminator, an hRNU5A-l terminator, an mRNU5g terminator, or an hRNU12 terminator. Attorney Docket No. 062692-509001 WO

[0178] In some embodiments, a terminator includes an mRNUl-2 terminator, an mRNU5A-l terminator, an hRNU5g terminator, or an mRNU12 terminator.

[0179]

[0120] In some embodiments, a system includes a truncated snRNA terminator. The terminator may be operably linked to a transcribable sequence. The truncated snRNA terminator may be or include a functional fragment of a terminator sequence. The truncated snRNA terminator may be or include a functional fragment of a RNU1-2 terminator sequence. The truncated snRNA terminator may be or include a functional fragment of a RNU5A-1 terminator sequence. The truncated snRNA terminator may be or include a functional fragment of a RNU5g terminator sequence. The truncated snRNA terminator may be or include a functional fragment of a RNU12 terminator sequence.

[0180]

[0121] A terminator may be or include an RNU1-2 terminator. The terminator may be or include an hRNUl-2 terminator. The terminator may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an hRNUl-2 terminator sequence provided herein. The terminator may be or include a functional fragment of a terminator. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated hRNUl-2 terminator sequence provided herein.

[0181]

[0122] A terminator may be or include an RNU5A-1 terminator. The terminator may be or include an hRNU5A-l terminator. The terminator may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an hRNU5A-l terminator sequence provided herein. The terminator may be or include a functional fragment of a terminator. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated hRNU5A-l terminator sequence provided herein.

[0182]

[0123] A terminator may be or include an RNU5g terminator. The terminator may be or include an mRNU5g terminator. The terminator may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an mRNU5g terminator sequence provided herein. The terminator may be or include a functional fragment of a terminator. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated mRNU5g terminator sequence provided herein.

[0183]

[0124] A terminator may be or include an RNU12 terminator. The terminator may be or include an hRNU12 terminator. The terminator may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an hRNU12 terminator sequence provided herein. The terminator may be or include a functional fragment of a terminator. The functional fragment may include or consist of a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a truncated hRNU12 terminator sequence provided herein. Attorney Docket No. 062692-509001 WO

[0184]

[0125] In some embodiments, the snRNA terminator or the RNU1-2 terminator sequence comprises a human RNU1-2 terminator (hRNUl-2) sequence. In some embodiments, the snRNA terminator or the hRNUl-2 terminator sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a hRNUl-2 terminator sequence herein, or a functional fragment thereof. In some embodiments, the snRNA terminator or the RNU5A-1 terminator sequence comprises a human RNU5A-1 terminator (hRNU5A-l) sequence. In some embodiments, the snRNA terminator or the hRNU5A-l terminator is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an hRNU5A-l terminator sequence herein, or a functional fragment thereof. In some embodiments, the snRNA terminator or the RNU5g terminator sequence comprises a mouse RNU5g terminator (mRNU5g) sequence. In some embodiments, the snRNA terminator or the mRNU5g terminator sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an mRNU5g terminator sequence herein, or a functional fragment thereof. In some embodiments, the snRNA terminator or the RNU12 terminator sequence comprises a human RNU12 terminator (hRNU12) sequence. In some embodiments, the snRNA terminator or the hRNU12 terminator sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an hRNU12 terminator sequence herein, or a functional fragment thereof.

[0185]

[0126] A recombinant terminator may be or include a functional fragment. In some embodiments, a terminator is or includes a functional terminator fragment. In some embodiments, the functional fragment is about 50 base pairs (bp), about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp, or a range defined by any 2 of the aforementioned bp lengths. In some embodiments, the functional fragment is less than 75 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, or less than 500 bp. In some embodiments, the functional fragment is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, or at least 450 bp. In some embodiments, the functional fragment is a 5’ fragment. In some embodiments, the functional fragment is a 3’ fragment. In some embodiments, the functional fragment is an internal fragment.

[0186]

[0127] An expression system may include a recombinant small nuclear RNA (snRNA) terminator. The snRNA terminator may be operably linked to a transcribable sequence. The snRNA terminator may include multiple proximal regulatory sequence elements (PSEs) (e.g., 2, 3, 4, or 5 PSEs). The snRNA terminator may include multiple distal regulatory sequence elements (DSEs) (e.g. 2, 3, 4, or 5 DSEs). The snRNA terminator may include multiple 3’ boxes (e.g., 2, 3, 4, or 5 3’ boxes). Attorney Docket No. 062692-509001 WO

[0187]

[0128] An expression system may include a recombinant small nuclear RNA (snRNA) terminator operably linked to a transcribable sequence. The terminator may include a Kozak sequence. The terminator may exclude a Kozak sequence. The terminator may include a Kozak sequence deletion. In some embodiments, the Kozak sequence comprises 5’-GCCAACCATG-3’.

[0188]

[0129] In some embodiments, the terminator sequence may include a 3’ terminator sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the promoter sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the ESS nucleic acid sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the target nucleic acid sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the Sm binding site. In some embodiments, the terminator sequence is 3’ or downstream relative to the hairpin sequence. In some embodiments, the terminator sequence is 3’ or downstream relative to the promoter sequence, the ESS nucleic acid sequence, the target nucleic acid sequence, the Sm binding site, or the hairpin sequence.

[0189]

[0130] In some embodiments, the terminator is directly downstream of the open reading frame (ORF). In some embodiments, the terminator is 5 bps downstream of the ORF. In some embodiments, the terminator is 10 bps downstream of the ORF. In some embodiments, the terminator is 15 bps downstream of the ORF. In some embodiments, the terminator is 20 bps downstream of the ORF. In some embodiments, the terminator is 25 bps downstream of the ORF. In some embodiments, the terminator is 30 bps downstream of the ORF. In some embodiments, the terminator is 35 bps downstream of the ORF. In some embodiments, the terminator is 40 bps downstream of the ORF. In some embodiments, the terminator is 45 bps downstream of the ORF. In some embodiments, the terminator is 50 bps downstream of the ORF. In some embodiments, the terminator is 55 bps downstream of the ORF. In some embodiments, the terminator is 60 bps downstream of the ORF. In some embodiments, the terminator is 65 bps downstream of the ORF. In some embodiments, the terminator is 70 bps downstream of the ORF. In some embodiments, the terminator is 75 bps downstream of the ORF. In some embodiments, the terminator is 80 bps downstream of the ORF. In some embodiments, the terminator is 85 bps downstream of the ORF. In some embodiments, the terminator is 90 bps downstream of the ORF. In some embodiments, the terminator is 95 bps downstream of the ORF. In some embodiments, the terminator is 100 bps downstream of the ORF. In some embodiments, the terminator is 200 bps downstream of the ORF. In some embodiments, the terminator is 300 bps downstream of the ORF. In some embodiments, the terminator is 400 bps downstream of the ORF. In some embodiments, the terminator is 500 bps downstream of the ORF. In some embodiments, the terminator is 600 bps downstream of the ORF. In some embodiments, the terminator is 700 bps downstream of the ORF. In some embodiments, the terminator is 800 bps downstream of the ORF. In some embodiments, the terminator is 900 bps downstream of the ORF. In some embodiments, the terminator is 1000 bps downstream of the ORF. Attorney Docket No. 062692-509001 WO

[0190] Compositions

[0191]

[0131] Disclosed herein, in some embodiments, are compositions. The composition may be a pharmaceutical composition. The composition may include a delivery agent such as a viral delivery agent. The composition may include RNAs such as recombinant or modified U7 snRNA sequences described herein, or an expression construct.

[0192]

[0132] Described herein, in some embodiments, the components of the system may be combined together within a single nucleic acid. In some embodiments, the components are separated among multiple nucleic acids.

[0193]

[0133] In some embodiments, a pharmaceutical composition comprising the system and a pharmaceutically acceptable carrier is described. A composition may include a carrier such as a pharmaceutically acceptable carrier. Examples of carriers may include a solution such as water, a buffer, or saline, or a lipid composition.

[0194]

[0134] Some embodiments relate to or include a virus. In some embodiments, a virus may include the nucleic acid system. In some embodiments, the virus is a parvovirus. An example of a parvovirus may include a dependoparvo virus. An example of a dependoparvo virus may include an adeno-associated virus (AAV). In some embodiments, the virus may be an AAV. In some embodiments, the AAV is a self-complementary AAV. In some embodiments, the AAV is a singlestrand AAV. In some embodiments, the AAV may be serotype AAV1. In some embodiments, the AAV may be serotype AAV2. In some embodiments, the AAV may be serotype AAV4. In some embodiments, the AAV may be serotype AAV5. In some embodiments, the AAV may be serotype AAV6. In some embodiments, the AAV may be serotype AAV7. In some embodiments, the AAV may be serotype AAV8. In some embodiments, the AAV may be serotype AAV9.

[0195]

[0135] In some embodiments, the AAV may express one U7 cassette. Gene cassettes may include small mobile elements, consisting of a single gene and a recombination site, which may be integrated into larger elements called integrons. Several gene cassettes can be inserted into the same integrin forming a tandem array where cassettes can be expressed together. In some embodiments, the AAV may express an array of two U7 cassettes. In some embodiments, the AAV may express an array of three U7 cassettes. In some embodiments, the AAV may express an array of four U7 cassettes. In some embodiments, the AAV may express an array of five U7 cassettes. In some embodiments, the AAV may express at least one U7 cassette. In some embodiments, the AAV may express an array of at least two U7 cassettes. In some embodiments, the AAV may express an array of at least three U7 cassettes. In some embodiments, the AAV may express an array of at least four U7 cassettes. In some embodiments, the AAV may express an array of at least five U7 cassettes. In some embodiments, the AAV may express at most one U7 cassette. In some embodiments, the AAV may express an array of at most two U7 cassettes. In some embodiments, the AAV may express an array of at most three U7 cassettes. In some embodiments, the AAV may express an array of at most four U7 cassettes. In some embodiments, the AAV may express an array of at most five U7 cassettes. Attorney Docket No. 062692-509001 WO

[0196]

[0136] Disclosed herein, in some embodiments, are recombinant viruses. A virus such as a recombinant virus may include a viral protein. A virus may include a viral envelope. A virus may include a nucleic acid expression system. The system may include a promoter. The promoter may be operably linked to a transcribable sequence. The system may include a terminator. The terminator may be operably linked to a transcribable sequence. The promoter may include a U12 small nuclear RNA (RNU12) promoter sequence. The terminator may include a U1 small nuclear RNA 2 (RNU1-2) terminator sequence. The promoter may include a U3B small nuclear RNA 1 (RNU3bl) promoter sequence. The terminator may include a U5A small nuclear 1 RNA (RNU5A-1) terminator sequence. The promoter may include a U2 small nuclear RNA 10 (RNU2-10) promoter sequence. The terminator may include a U5g small nuclear RNA (RNU5g) terminator sequence. The terminator may include an RNU12 terminator sequence. In some embodiments, the virus is an adeno-associated virus.

[0197]

[0137] In some embodiments, a cell comprises the system. In some embodiments, the cell is a brain cell. In some embodiments, the cell is a neural cell. In some embodiments, the cell is a neuron.

[0198] Methods of Use

[0199]

[0138] Described herein, in some embodiments, are methods. The method may include administering a composition or system described herein. The method of administering can comprise retroorbital administration. The method may include delivering a composition or system described herein in a cell. The method of delivery can comprise injection (e.g., retroorbital injection). The method may include expressing a composition or system described herein in a cell. The cell may be in vivo (e.g., in a living body). The cell may be in vitro. The method may include a method of treatment. The method may include modifying splicing of a target nucleic acid such as a SCN2A RNA. The method may be performed on a subject, or on a cell such as a cell of a subject.

[0200]

[0139] Described herein, in some embodiments, is a method comprising administering a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a virus. The composition or virus may be modified. The composition or virus may be recombinant. In some embodiments, the pharmaceutical composition comprises a virus that is an adeno-associated virus (AAV). In some embodiments, the virus comprises a promoter, an exonic splicing silencer sequence, a U7 target sequence, a smOPT, a U7 3’ hairpin structure, and a 3’ terminal sequence. Described herein, in some embodiments, is a method of administering a pharmaceutical composition or virus comprised of the nucleic acid system.

[0201]

[0140] Some embodiments relate to or include a treatment method. A method (such as a treatment method) may include administering a nucleic acid expression system to a subject such as a mammalian subject. The nucleic acid expression system may include a promoter. The promoter may be operably linked to a transcribable sequence. The nucleic acid expression system may include a terminator. The terminator may be operably linked to a transcribable sequence. The promoter may include a U12 small nuclear RNA (RNU12) promoter sequence. The terminator may include a U1 Attorney Docket No. 062692-509001 WO small nuclear RNA 2 (RNU1-2) terminator sequence. The promoter may include a U3B small nuclear RNA 1 (RNU3bl) promoter sequence. The terminator may include a U5A small nuclear 1 RNA (RNU5A-1) terminator sequence. The promoter may include a U2 small nuclear RNA 10 (RNU2-10) promoter sequence. The terminator may include a U5g small nuclear RNA (RNU5g) terminator. The terminator may include an RNU12 terminator sequence.

[0202]

[0141] Some embodiments include methods of producing a ribonucleic acid (RNA), comprising contacting a cell with a virus or system described herein.

[0203]

[0142] The method may be used to treat or modify splicing in a subject. The method may include administering a composition to a subject. The method may be used to treat a cell or modify splicing in a cell. The method may include administering a composition to a cell. The cell may be a brain cell. The cell may be a neural cell. The cell may be a neuron.

[0204]

[0143] In some embodiments, the cell is in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject may have one mutated copy of SCN2A DNA per cell. In some embodiments, the subject may have two mutated copies of SCN2A DNA per cell. In some embodiments, the subject may have no mutated copies of SCN2A. A mutated copy of SCN2A may lead to splicing that includes an alternatively spliced exon of the SCN2A. A mutated copy of SCN2A may result in nonsense-mediated decay (NMD) of a SCN2A mRNA.

[0205] Modifying Splicing

[0206]

[0144] Described herein, in some embodiments, is a method for modifying splicing. The method may include modifying splicing in a subject. The method may include modifying splicing in a cell. In some embodiments, the method may include contacting a pre-mRNA with a system or composition herein. In some embodiments, the pre-mRNA may encode SCN2A. In some embodiments, the method may include contacting a pre-mRNA encoding SCN2A with a recombinant nucleic acid sequence that induces exclusion of exon 13N from a mature mRNA generated by the pre-mRNA. Described herein, in some embodiments, is a method of modifying splicing comprised of contacting a pre-mRNA encoding SCN2A with a recombinant nucleic acid sequence that induces exclusion of exon 13N from a mature mRNA generated by the pre-mRNA.

[0207]

[0145] In some embodiments, the pre-mRNA is in a cell. In some embodiments, the pre-mRNA is in a neural cell. The cell may be a brain cell. The cell may be a neuron.

[0208]

[0146] In some embodiments, the subject has a disorder. The disorder may include a neurodevelopmental disorder. For example, some embodiments include modifying splicing in cells of a subject that has a neurodevelopmental disorder. In some embodiments, the subject is at risk of having the neurodevelopmental disorder.

[0209]

[0147] Described herein, in some embodiments, is a method of preventing, reducing or inhibiting nonsense-mediated decay (NMD). The NMD may be prevented. The NMD may be reduced. The Attorney Docket No. 062692-509001 WO

[0210] NMD may be inhibited. The NMD may be prevented, reduced, or inhibited in a subject. The NMD may be prevented, reduced, or inhibited in a cell of a subject. The NMD may be prevented, reduced, or inhibited with regard to a SCN2A transcript. Some embodiments include preventing, reducing or inhibiting nonsense-mediated decay (NMD) by contacting a SCN2A pre-mRNA with a composition or system described herein, such as a modified U7 snRNA. Some embodiments include preventing, reducing or inhibiting nonsense-mediated decay (NMD) by administering composition or system described herein to a subject. The prevention, reduction, or inhibition may be relative to a baseline or control.

[0211]

[0148] Some embodiments include reducing or inhibiting NMD in cells (e.g., cells of a subject) by at least 10%. Some embodiments include reducing or inhibiting NMD in cells by at least 20%. Some embodiments include reducing or inhibiting NMD in cells by at least 30%. Some embodiments include reducing or inhibiting NMD in cells by at least 40%. Some embodiments include reducing or inhibiting NMD in cells by at least 50%. Some embodiments include reducing or inhibiting NMD in cells by at least 60%. Some embodiments include reducing or inhibiting NMD in cells by at least 70%. Some embodiments include reducing or inhibiting NMD in cells by at least 80%. Some embodiments include reducing or inhibiting NMD in cells by at least 90%. Some embodiments include reducing or inhibiting NMD in cells by less than 10%. Some embodiments include reducing or inhibiting NMD in cells by less than 20%. Some embodiments include reducing or inhibiting NMD in cells by less than 30%. Some embodiments include reducing or inhibiting NMD in cells by less than 40%. Some embodiments include reducing or inhibiting NMD in cells by less than 50%. Some embodiments include reducing or inhibiting NMD in cells by less than 60%. Some embodiments include reducing or inhibiting NMD in cells by less than 70%. Some embodiments include reducing or inhibiting NMD in cells by less than 80%. Some embodiments include reducing or inhibiting NMD in cells by less than 90%. Some embodiments include reducing or inhibiting NMD in cells by less than 95%. Some embodiments include reducing or inhibiting NMD in cells by less than 100%. Some embodiments include a range of any of the above percentages.

[0212]

[0149] Described herein, in some embodiments, is a method of administering to the subject a composition that silences or reduces said splicing, the composition comprising the system, virus, or composition described above. In some embodiments, the method comprises administering to the subject a composition that silences the splicing. In some embodiments, the method comprises administering to the subject a composition that reduces the splicing. For example, silencing splicing of exon 13N may prevent exon 13N from being included in a mature SCN2A mRNA.

[0213]

[0150] In some embodiments, the method may increase an amount of a productive isoform of SCN2A. In some embodiments, the increase in the amount of a productive isoform of SCN2A is relative to a control. In some embodiments, the increase in the amount of a productive isoform of SCN2A is relative to a baseline amount of said productive isoform. In some embodiments, the method Attorney Docket No. 062692-509001 WO increases an amount of a productive isoform of SCN2A, relative to a control or baseline amount of said productive isoform.

[0214]

[0151] In some embodiments, the method may increase an amount of a NaV1.2 channel or protein (e.g., SCN2A protein). In some embodiments, the method may increase an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a control. In some embodiments, the method may increase an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a baseline amount of the NaV1.2 channel or protein. In some embodiments, the method increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein), relative to a control or baseline amount of the NaV1.2 channel or protein.

[0215]

[0152] In some embodiments, the method improves sodium transport. In some embodiments, the method improves sodium transport relative to a control. In some embodiments, the method improves sodium transport relative to a baseline amount. In some embodiments, the method improves sodium transport, relative to a control or baseline amount. The improvement may be by at least 10%.

[0216]

[0153] Some embodiments relate to or include a method of reducing an amount of a nonproductive SCN2A transcript. The non-productive SCN2A transcript may include a mature SCN2A mRNA that includes exon 13N. The amount of non-productive SCN2A transcript may be reduced in a cell or subject. The amount of non-productive SCN2A transcript may be reduced relative to a baseline measurement. The amount of non-productive SCN2A transcript may be reduced relative to a control measurement. The amount of non-productive SCN2A transcript may be reduced by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, or by at least 90%. In some embodiments, the amount of non-productive SCN2A transcript is reduced by less than 10%, by less than 20%, by less than 30%, by less than 40%, by less than 50%, by less than 60%, by less than 70%, by less than 80%, by less than 90%, or by less than 100%. The amount of non-productive SCN2A transcript may be reduced by a range of percentages herein.

[0217]

[0154] The amount of non-productive SCN2A transcript may be measured in a sample of a subject. The sample may include a biofluid such as blood, serum, plasma, or cerebrospinal fluid, or may include a tissue sample such as neural or brain tissue. The baseline amount of non-productive SCN2A transcript may be measured in a baseline sample obtained before treatment of the subject (e.g., before administration of a composition herein to the subject). The amount of productive SCN2A transcript may be measured with an assay method such as a PCR assay. Examples of PCR assays may include quantitative PCR (qPCR) or reverse transcription quantitative PCR (RT-qPCR). The amount of non-productive SCN2A transcript may be normalized to a control, such as a measurement of a housekeeping mRNA.

[0218]

[0155] Some embodiments relate to or include a method of increasing an amount of a productive SCN2A transcript. The productive SCN2A transcript may include a mature SCN2A mRNA that does not include exon 13N. The amount of productive SCN2A transcript may be increased in a cell or subject. The amount of productive SCN2A transcript may be increased relative to a baseline Attorney Docket No. 062692-509001 WO measurement. The amount of productive SCN2A transcript may be increased relative to a control measurement. The amount of productive SCN2A transcript may be increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 225%, or by at least 250%. In some embodiments, the amount of productive SCN2A transcript is increased by less than 10%, by less than 20%, by less than 30%, by less than 40%, by less than 50%, by less than 60%, by less than 70%, by less than 80%, by less than 90%, by less than 100%, by less than 125%, by less than 150%, by less than 175%, by less than 200%, by less than 225%, by less than 250%, by less than 275%, by less than 300%, by less than 350%, or by less than 400%. The amount of productive SCN2A transcript may be increased by a range of percentages herein.

[0219]

[0156] The amount of productive SCN2A transcript may be measured in a sample of a subject. The sample may include a biofluid such as blood, serum, plasma, or cerebrospinal fluid, or may include a tissue sample such as neural or brain tissue. The baseline amount of productive SCN2A transcript may be measured in a baseline sample obtained before treatment of the subject. The amount of productive SCN2A transcript may be measured with an assay method such as a PCR assay. The amount of productive SCN2A transcript may be normalized to a control, such as a measurement of a housekeeping mRNA.

[0220]

[0157] Some embodiments relate to or include a method of increasing an amount of NaV1.2 protein. The amount of NaV1.2 protein may be increased in a cell or subject. The amount of NaV1.2 protein may be increased relative to a baseline measurement. The amount of NaV1.2 protein may be increased relative to a control measurement. The amount of NaV 1.2 protein may be increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by 100%, by at least 125%, by at least 150%, or by at least 160%. In some embodiments, the amount of NaV1.2 protein is increased by less than 10%, by less than 20%, by less than 30%, by less than 40%, by less than 50%, by less than 60%, by less than 70%, by less than 80%, by less than 90%, by less than 100%, by less than 125%, by less than 150%, by less than 160%, by less than 175%, or by less than 200%. The amount of NaV1.2 protein may be increased by a range of percentages herein. In some embodiments, the amount of NaV1.2 protein may be increased by about 0% to 20% relative to a control measurement. In some embodiments, the amount of NaV1.2 protein may be increased by about 0% to 10% relative to a control measurement. In some embodiments, the amount of NaV1.2 protein may be increased by about 0% to 5% relative to a control measurement. In some embodiments, the amount of NaV1.2 protein may be increased by about 20% relative to a control measurement.

[0221]

[0158] The amount of NaV1.2 protein may be measured in a sample of a subject. The sample may include a biofluid such as blood, serum, plasma, or cerebrospinal fluid, or may include a tissue sample such as neural or brain tissue. The baseline amount of NaV1.2 protein may be measured in a Attorney Docket No. 062692-509001 WO baseline sample obtained before treatment of the subject. The amount of NaV1.2 protein may be measured with an assay method such as an immunoblot. The amount of NaV1.2 protein may be normalized to a control, such as a measurement of total protein or of a housekeeping protein.

[0222] Treating a Disorder

[0223]

[0159] Described herein, in some embodiments, is a method for treating a disorder in a subject in need thereof. In some embodiments, the subject has the disorder. In some embodiments, the subject is identified as having the disorder. In some embodiments, the subject is at risk of having the disorder. In some embodiments, the subject is identified as at risk of having the disorder. The treatment may have a prophylactic effect. The method may include any aspect of another method described herein, such as modifying splicing, reducing an amount of a non-productive SCN2A transcript relative to a baseline or control measurement, increasing an amount of a non-productive SCN2A transcript relative to a baseline or control measurement, or increasing an amount of NaV1.2 protein relative to a baseline or control measurement.

[0224]

[0160] The disorder may be or include a genetic disorder. In some embodiments, the subject has or is at risk of having a genetic disorder. The disorder may be or include a neurodevelopmental disorder. In some embodiments, the subject has or is at risk of having a neurodevelopmental disorder. In some embodiments, the neurodevelopmental disorder comprises intellectual disability, autistic features, or epilepsy. The disorder may include an intellectual disability. The disorder may include autism or an autistic feature.

[0225]

[0161] Described herein, in some embodiments, is a method for preventing a disorder in a subject in need thereof. Described herein, in some embodiments, is a method for treating a disorder in a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of a synthetic composition that may silence splicing of an alternatively spliced region of a ribonucleic acid (RNA). In some embodiments, the method comprises administering a therapeutically effective amount of a synthetic composition that may reduce splicing of an alternatively spliced region of RNA. In some embodiments, the method comprises administering a therapeutically effective amount of a synthetic composition that may silence splicing of an alternatively spliced region of RNA encoding SCN2A. In some embodiments, the method comprises administering a therapeutically effective amount of a synthetic composition that may reduce splicing of an alternatively spliced region of RNA encoding sodium channel protein type 2 subunit alpha (SCN2A). Described herein, in some embodiments, is a method of treating or preventing epilepsy in a subject in need thereof, comprised of administering a therapeutically effective amount of a synthetic composition that silences or reduces splicing of an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the alternatively spliced region may include an exon. In some embodiments, the alternatively spliced region may include exon 13N of the RNA. Attorney Docket No. 062692-509001 WO

[0226]

[0162] Described herein, in some embodiments, is a method of treating or preventing a neurodevelopmental disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant U7 small nuclear RNA (snRNA) composition that silences or reduces splicing of an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A). In some embodiments, the neurodevelopmental disorder comprises intellectual disability, autistic features, or epilepsy.

[0227]

[0163] In some embodiments, the administration may increase the amount of a productive isoform of SCN2A in the subject. In some embodiments, the administration may increase the amount of a productive isoform of SCN2A in the subject relative to a control. In some embodiments, the administration may increase the amount of a productive isoform of SCN2A in the subject relative to a baseline amount of said productive isoform. In some embodiments, the method increases an amount of a productive isoform of SCN2A in the subject, relative to a control or baseline amount of said productive isoform.

[0228]

[0164] In some embodiments, the administration may increase the amount of a NaV1.2 channel or protein (e.g. SCN2A protein) in the subject. In some embodiments, the administration may increase the amount of a NaV1.2 channel or protein (e.g. SCN2A protein) in the subject relative to a control. In some embodiments, the administration may increase the amount of a NaV1.2 channel or protein (e.g., SCN2A protein) in the subject relative to a baseline amount of the NaV1.2 channel or protein. In some embodiments, the administration increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein) in the subject, relative to a control or baseline amount of the NaV1.2 channel or protein.

[0229]

[0165] In some embodiments, the administration may improve sodium transport in the subject. In some embodiments, the administration may improve sodium transport in the subject relative to a control. In some embodiments, the administration may improve sodium transport in the subject relative to a baseline amount of sodium transport. In some embodiments, the administration improves sodium transport in the subject, relative to a control or baseline amount.

[0230]

[0166] The disorder may include epilepsy. The disorder may include a seizure. In some embodiments, the subject has epilepsy. In some embodiments, the subject is at risk of having epilepsy. Epilepsy is a chronic noncommunicable disease of the brain that affects around 50 million people worldwide. Epilepsy can be characterized by recurrent seizures, which are brief episodes of involuntary movement that may involve a part of the body (partial) or the entire body (generalized) and are sometimes accompanied by loss of consciousness and control of bowel or bladder function. In some embodiments, the subject may suffer from tonic-clonic seizures. In some embodiments, the subject may suffer from myoclonic seizures. In some embodiments, the subject may suffer from atypical absence seizures. In some embodiments, the subject may suffer from atonic seizures. In some embodiments, the subject may suffer from focal aware or impaired awareness seizures. In some embodiments, the subject may suffer from tonic seizures. In some embodiments, the subject may Attorney Docket No. 062692-509001 WO suffer from non-convulsive status epilepticus. In some embodiments, the subject may be at risk of having tonic-clonic seizures. In some embodiments, the subject is at risk of having myoclonic seizures. In some embodiments, the subject may be at risk of having atypical absence seizures. In some embodiments, the subject may be at risk of having atonic seizures. In some embodiments, the subject may be at risk of having focal aware or impaired awareness seizures. In some embodiments, the subject may be at risk of having tonic seizures. In some embodiments, the subject may be at risk of having non-convulsive status epilepticus seizures.

[0231]

[0167] In some embodiments, the administration prevents the subject from having seizures. In some embodiments, the administration reduces the amount of the seizures of the subject relative to a baseline amount. In some embodiments, the administration reduces the severity of the seizures of the subject relative to a baseline severity. In some embodiments, the administration prevents the subject from having seizures, or reduces an amount or severity of the seizures of the subject relative to a baseline amount or severity.

[0232] Definitions

[0233]

[0168] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0234]

[0169] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0235]

[0170] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value. The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and Attorney Docket No. 062692-509001 WO qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0236]

[0171] Some embodiments relate to a small nuclear RNA (snRNA). An snRNA may include a class of small RNA molecules found within splicing speckles or Cajal bodies of a eukaryotic cell nucleus. A length of an unmodified snRNA may average about 150 nucleotides. An snRNA may be transcribed by RNA polymerase II or RNA polymerase III. An snRNA may function in the processing of pre-messenger RNA (hnRNA) in the nucleus. Any of these aspects may be modified or missing in a modified or engineered snRNA. An snRNA may associate with a protein or set of proteins to form a complex. The complex may be referred to as a small nuclear ribonucleoprotein (snRNP). Some examples of human snRNA components of such complexes may include: U1 spliceosomal RNA, U2 spliceosomal RNA, U4 spliceosomal RNA, U5 spliceosomal RNA, or U6 spliceosomal RNA. An snRNA may have a high uridine content.

[0237]

[0172] Some embodiments relate to a U7 snRNA. A U7 snRNA may include an RNA molecule and a component of a small nuclear ribonucleoprotein complex (U7 snRNP). The U7 snRNA may affect histone pre-mRNA processing. The U7 snRNA may be modified or engineered. In some embodiments, the modified or engineered U7 snRNA does not affect histone pre-mRNA processing, or has little effect on such. In some embodiments, a U7 snRNA has a 5’ end that binds an HDE (histone downstream element), a conserved purine-rich region, located 15 nucleotides downstream a histone mRNA cleavage site. Any of these aspects may be modified or missing in a modified or engineered U7 snRNA. Binding of an HDE region by a U7 snRNA, through complementary basepairing, may affect recruitment of cleavage factors during histone pre-mRNA processing.

[0238]

[0173] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. A biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0239]

[0174] A percent sequence identity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the % sequence identity. A sequence Attorney Docket No. 062692-509001 WO identity may include a sequence identity to a reverse complement. In determining a sequence identity, thymine (T) and uracil (U) may be interchangeable. T and U may be interchangeable when describing an oligonucleotide. In some embodiments, Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T. Where a T is provided as a part of DNA, a U may be envisaged as part of an RNA, and vice versa.

[0240]

[0175] Any discrepancies between the written description and a sequence listing submitted herewith may typically be resolved in favor of the written description.

[0241]

[0176] As used herein, the terms “treatment” or “treating” may be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit may be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect may include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0242]

[0177] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0243] EXAMPLES

[0244]

[0178] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0245] Example 1: Therapeutic Regulation of SCN2A Splicing by Engineered U7 snRNAs

[0246]

[0179] The presence of SCN2A splice isoforms that include exon 13N was confirmed in mouse Neuro-2a cells, differentiated human cortical progenitors (ReNcell CX), and human brain RNA, and it was confirmed to be subject to NMD via treatment of Neuro-2a cells with the NMD inhibitor cycloheximide (FIG. 1). Here it was hypothesized that suppression of splicing into exon 13N could increase splicing of the normal productive SCN2A splice isoform, leading to increased levels of NaV1.2.

[0247]

[0180] U7 snRNA is an endogenous cell cycle-regulated small RNA that modulates splicing of histone mRNAs. Modification of the U7 backbone to exclude binding of histone-specific splicing factors and recruit splicing proteins allows the use of U7 as a targeted splicing modulator. Through a Attorney Docket No. 062692-509001 WO combination of targeting position within the mRNA of interest and the addition of 5’ exonic splicing silencer (ESS) sequences, U7 can be used to engage endogenous mRNAs in a sequence-specific manner and subsequently up-regulate or down-regulate the splicing of specific exons5. A functional U7 cassette consists of, in the following order from 5’ to 3’: 1) a promoter, 2) a splicing silencer, 3) a targeting sequence complementary to a region of a target mRNA, 4) an smOPT sequence, 5) a hairpin sequence, and 6) a 3’ termination signal.

[0248] Approach

[0249]

[0181] An adeno-associated virus (AAV)-based gene therapy was engineered for the treatment of SCN2A haploinsufficiency utilizing an engineered U7 cassette to target and prevent splicing of NMD- inducing alternative exon, 13N. The gene therapy vector included the following components, either as a single expression cassette or an arrayed expression cassette. Examples of some relevant sequences for U7 core components and regulatory elements are provided in Table 1 and Table 5):

[0250] 1. Promoter o Mouse or human U7, mulal, HUI-1, or U7 with DSE and PSE replaced with those from HUI-1 and mulal, respectively

[0251] 2. Exonic splicing silencer (ESS) sequence

[0252] 3. U7 target sequence

[0253] 4. smOPT

[0254] 5. U7 3’ hairpin structure

[0255] 6. 3’ Terminator Sequence o Mouse U7 or human Ul-1 (HUI)

[0256] Results

[0257]

[0182] A goal was to validate our U7-based splice modulation approach for increasing Scn2a levels by performing a screen with a luciferase-based splice reporter whose function is tied to alternative splicing between exons 13, 13N, and 14 of Scn2a in mouse Neuro-2a cells. The alternatively spliced NMD exon present in humans is 93% conserved in mice (125 / 135 identity) and the region encompassing the exon, including 50bp upstream and 60bp downstream, is also highly conserved (219 / 245 identical bases; FIG. 2). A library was designed to target the entire NMD exon and flanking regions that consisted of 47 unique antisense sequences of 20, 24, 26, or 30 nucleotides in length. The “antisense” sequences may also be referred to as “target” or “targeting” sequences, since a target sequence typically is a reverse complement or otherwise antisense to a target RNA. Targeting sequences were paired with multiple exonic splicing silencer sequences (Table 2). We then transfected Neuro-2a cells and analyzed the suppression of NMD exon inclusion via a dual luciferase assay where suppression of NMD exon inclusion results in increased firefly luciferase signal intensity (normalized to renilla luciferase). Using this method, we identified 10 antisense / ESS combinations Attorney Docket No. 062692-509001 WO suitable for further validation (FIG. 3, Table 2, Table 3). Inclusion of the NMD exon results in nonsense mediated decay and low luciferase expression. Suppression of the NMD exon promotes inclusion of the in-frame exon 14 fragment and results in increased luciferase expression.

[0258]

[0183] The 10 candidate U7 cassettes were validated by assessing their ability to suppress endogenous Scn2a NMD exon inclusion in Neuro-2a cells. End-point RT-PCR showed that all 10 constructs were capable of near-complete suppression of NMD exon inclusion, as well as upregulation of productive Scn2a isoform production (FIG. 4). To confirm the ability of these candidates to suppress NMD exon inclusion in human cells, the 3 most conserved sequences were cloned into AAV vectors in a 3X array, and differentiated ReNcell CX cultures were transduced. After culturing transduced cells for 10 days, protein was purified and NaV1.2 levels were analyzed, which showed that treatment with candidate U7 constructs increased NaV1.2 expression by 41-63% (FIG. 5).

[0259]

[0184] Next, with the goal of identifying clinical candidates with cross-species activity, a library was generated of U7 constructs with perfect complementarity to the human SCN2A NMD exon 13N. The library was screened for increased luciferase activity with the mouse Scn2a luciferase-based splice reporter in Neuro-2A cells. This approach was used for several reasons. First, experiments using the mouse library showed that screening for hits in Neuro-2A cells can identify high-confidence candidates capable of suppressing NMD exon inclusion in mouse and human cells. Second, given that a bulk of development and proof-of-concept work can be carried out in mice, candidates with high activity in mouse cells were prioritized. By first identifying human sequence-specific candidates that have high activity in mouse cells, candidates with cross-species activity may then be quickly identified. Screening results in Neuro-2A cells with the human sequence-specific library are shown in Table 4 and FIG. 6. These data yielded several high-confidence hits capable of increasing reporter expression (e.g., by greater than 50% shown by circles with centers above the dotted line in the figure).

[0260]

[0185] Some constructs included an exonic splicing silencer (ESS) sequence, while others did not. The ESS did not appear as an obligate requirement for U7 activity. Those lacking an ESS that work well may bind or block sequence motifs that promote splicing. Those that work with an ESS may also block such motifs, and function further by positioning their artificial ESS motifs in a useful context near a splice junction.

[0261] Table 2. Sequences of exonic splicing silencers and mouse U7 antisense sequences tested in splice reporter screen and relative renilla-normalized luciferase levels. Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0262] Table 3. ESS and antisense sequences for candidates used for further validation in Neuro-2a cells

[0263] Table 4. Sequences of exonic splicing silencers and human U7 target sequences tested in splice reporter screen and relative renilla-normalized luciferase levels. Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0264] Example 2: Platform expansion and validation of promoter / terminator combinations driving U7 expression and U7-mediated splice modulation

[0265]

[0186] Combinations of promoter and terminator regions from mouse and human snRNAs were designed tested. Because some core promoter and terminator elements are sometimes not well-defined for all small RNAs across species, regions of ~500bp upstream and downstream of the coding regions of human and mouse small RNAs were selected for testing. A library of constructs, each encoding an engineered U7 snRNA targeting exon 13N of mouse Scn2a driven by one of 529 combinations of the chosen promoters and terminators was generated (Table 7A). Construct fragments in Table 7A were named sequentially in the order of a three-digit fragment ID number, a recombinant promoter sequence (SEQ ID NOs: 6 and 162-184 from Table 5), a U7 sequence (Table 7B), and finally a recombinant 3’ signal / terminator sequence (SEQ ID NOs: 10 and 185-207 from Table 5), and were constructed 5’ to 3’ in the same order.

[0266]

[0187] To screen for the ability of various promoters and terminators to drive high levels of functional U7 expression, a luciferase reporter with the following features was generated: a relatively weak ubiquitous promoter (Ubc-SV40pA), a Kozak sequence, a fragment of mouse Scn2a exon 13, a fragment of mouse Scn2a intron 13, mouse Scn2a exon 13N, another fragment of mouse Scn2a intron 13, and a fragment of mouse Scn2a exon 14 fused in-frame to firefly luciferase (Table 6, FIG. 7). Under normal conditions a significant fraction of transcripts may include exon 13N, which is out-of- frame and leads to nonsense-mediated decay, thus reducing luciferase levels. Successful suppression of exon 13N inclusion by U7 results in proper in-frame expression of luciferase, which can be read out via luciferase enzymatic activity.

[0267]

[0188] Neuro-2a cells were co-transfected with the luciferase reporter, a control plasmid expressing Renilla luciferase, and individual plasmids from the 529 combinations. The library was split into 6 sub-libraries and each plate was screened in triplicate. Multiple control plasmids were included on each plate for reference. Following 24 hours of culture, average firefly luciferase levels, normalized to renilla luciferase, were calculated for each construct and an ANOVA was run for each sub-library to identify promoter / terminator combinations capable of significantly suppressing exon 13N inclusion, a direct proxy for functional U7 expression. Across libraries, 324 / 529 promoter / terminator combinations resulted in a statistically significant increase in luciferase activity (Tables 8-13). The averages in Tables 8-13 were generated using 3 replicates each. In line with previous findings that functional combinations are not obvious, even when combining elements from the same gene and species, there were many instances where a given promoter was only functional Attorney Docket No. 062692-509001 WO with the terminator of a different gene or species, suggesting there are unknown sequence determinants required for functional U7 expression. Together, these studies identify a suite of promoter / terminator combinations capable of driving high levels of functional U7 expression and expand the toolkit of regulatory elements for use of U7-mediated splice modulation.

[0268] Table 5: Example Regulatory Elements Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0269] Table 6: Luciferase Reporter Attorney Docket No. 062692-509001 WO

[0270] Table 7A: Example Fragment Sequences in a Library of Constructs Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0271] Table 7B: Example U7 Sequences included in a Library of Constructs

[0272] Table 8: Library 1 Summary Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0273] Table 9: Library 2 Summary Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0274] SD, standard deviation.

[0275] Table 10: Library 3 Summary Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0276] SD, standard deviation.

[0277] Table 11: Library 4 Summary Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0278] SD, standard deviation.

[0279] Table 12: Library 5 Summary Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0280] SD, standard deviation.

[0281] Table 13: Library 6 Summary Attorney Docket No. 062692-509001 WO

[0282] SD, standard deviation.

[0283] Example 3 - Assessment of Modified Promoters and Terminators for Driving Expression of Functional Engineered snRNAs

[0284]

[0189] To improve understanding of promoter and terminator sequences useful for functional U7 expression, a series of deletion experiments was performed for some selected promoter / terminator pairs: hRNU12 / hRNUl-2, mRNU3bl / hRNU5Al, mRNU2-10 / mRNU5g, and mRNU2-10 / hRNU12. Additionally constructs pairing the full-length hRNU12 promoter with either the mRNUlal or mRNU7 terminators of varying lengths were tested to assess the core sequences useful for Ulal and U7 terminator function. For each promoter and terminator, a series of deletions were made beginning at the distal end, generating regulatory elements of the following lengths for testing: 500bp, 400bp, 300bp, 200bp, 150bp, lOObp, or 50bp. The shortened promoters and / or terminators were paired with their full-length partner regulatory element and used to drive expression of an ESS / antisense combination targeting the Scn2a luciferase reporter (Tables 14A-14C). Luciferase expression was then compared between the constructs with regulator elements of varying lengths and constructs completely lacking the regulatory element of interest. As shown in FIG. 8-13, the first lOObp was sufficient to provide full function of all tested terminators except mRNU7, which was unsuccessful without the first 200bp. In the case of hRNUl-2 and hRNU5A-l, the proximal 50bp sequence was sufficient to confer full function. For the tested promoters, the following sequences were useful for Attorney Docket No. 062692-509001 WO full activity: proximal 3OObp of hRNU12, proximal 200bp of mRNU2-10, proximal 300bp of mRNU3bl, proximal 300bp of mRNUlal, and the full 500bp of hRNUl-2 (FIG. 14-18).

[0285]

[0190] Functional U7 expression may be affected by sequence elements in the promoter and terminator which regulate both expression levels and proper U7 folding. Of particular usefulness are the PSE and DSE elements in the promoter and the 3’ box in the terminator. Thus, increasing the copy number of these elements within a given promoter and / or terminator can be used to increase functional U7 expression. To test this, a series of constructs encoding the same Scn2a-targeting ESS / antisense sequence under control of the mRNU2-10 / mRNU5g promoter / terminator pair were generated (Tables 14A-14C). Each construct had a combination of the following modifications: shortening of the mRNU2-10 promoter to 400bp, DSE duplication, PSE duplication, or 3’ box duplication. The constructs were transfected into Neuro-2A cells and their ability to both suppress Scn2a NMD isoform levels and increase productive Scn2a isoform levels was assessed. Duplication of the DSE, PSE, or 3’ box alone had no effect on functional U7 expression, but the combination of either PSE or DSE duplication along with 3’ box duplication was sufficient to increase productive transcript levels by -30% over the base plasmid (FIG. 19), indicating that these modifications were useful for increasing functional U7 expression.

[0286]

[0191] Safety is important for effective therapeutics. In the case of gene therapies, it is useful to eliminate unwanted transcripts and peptides from being expressed from the therapeutic construct. These products can arise from internal sequences that allow either transcriptional initiation or ribosomal binding and translation. The mRNU5g terminator is useful for regulating high levels of functional U7 expression but may contain a Kozak-ATG motif (e.g., 5’-GCCAACCATG-3’) that may serve as a scaffold for translation of an additional 52 amino acid peptide. In addition to potentially causing toxicity, this sequence could also affect stability of the U7 snRNA. To test this, the Kozak- ATG motif was removed from the mRNU5g terminator and a series of U7 constructs targeting Scn2a and driven by variants of the mRNU2-10 promoter were transfected in Neuro-2A cells (Tables 14A- 14C). Assessment of productive and NMD isoform levels of Scn2a was performed for each set of constructs, showing that removal of the Kozak-ATG motif did not affect function of the mRNU5g terminator and, in fact, slightly improved functional U7 expression across all constructs tested (FIG. 20). This indicates that such removal from the mRNU5g terminator can improve its function and expression of an engineered U7 snRNA operably connected to it.

[0287] Table 14A: Sequences Used in Experiments Using Modified Regulatory Elements Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0288] Table 14B: Example Promoters Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0289] Table 14C: Example Terminators Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO Attorney Docket No. 062692-509001 WO

[0290] Example 4 - Assessment of Constructs to Enhance Endogenous Productive Scn2a in Neuro-2a, U-87MG cells Promoters, and Scn2A Mutant Mice

[0291]

[0192] To address the differences between the human and mouse sequence of Scn2a and the exon 13N targeting region, an additional library was developed containing a mixture of sequences specific to the mouse and human mRNAs (Table 15A and Table 15B). All constructs in the library were designed with regulation by the mRNU3bl / hRNU5A-l promoter / terminator combination. To achieve a more accurate readout of their activity and assess their function in both species, the library was transfected into both mouse Neuro-2A and human U-87MG cells, both of which express Scn2a. After 72 hours of culture, RNA was purified and assessed for productive transcript levels via qRT- PCR. To identify candidates with the highest likelihood of working across species, all candidates were ranked based on their ability to increase productive Scn2a levels in both cell lines and then generated an aggregate score for each candidate (Table 15C). The top candidate in both cell lines was the only candidate whose target sequence is 100% conserved across species, suggesting that it targets a conserved regulatory element critical for the inclusion of exon 13N. To assess the ability of this candidate to increase productive Scn2a levels in vivo, AAV (PHP.eB) was delivered to Scn2a mutant mice at postnatal day 14 via retroorbital injection at a dose of 3el3vg / kg. Following 4 weeks of viral expression, whole brain lysates were prepared and Navi.2 protein levels were assayed via capillarybased analysis. A significant increase of about 20% in Navi.2 levels in animals dosed with this candidate (AS1) compared to saline-injected controls (Het) (FIG. 21). Together, these data validate the screening approach and the U7-based targeting of Scn2a exon 13N as a method for increasing Navi.2 levels.

[0292] Table 15A: ESS and Antisense Sequences for 20 Candidate Vectors Utilized for Screening of Endogenous Productive Scn2a in Neuro-2a and U-87MG cells Attorney Docket No. 062692-509001 WO

[0293] Table 15B: Promoters, Terminators, ESS and Antisense Sequences for 20 Candidate Vectors Utilized for Screening of Endogenous Productive Scn2a in Neuro-2a and U-87MG cells Attorney Docket No. 062692-509001 WO

[0294] Table 15C: Aggregate Ranking of Candidate Vectors Based on Relative Productive Scn2a levels in Neuro-2a and U-87MG cells

[0295] N2A, Neuro-2a cells; U-87, U-87MG cells.

[0296]

[0193] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of Attorney Docket No. 062692-509001 WO example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 062692-509001 WOCLAIMS1. A system for modifying nucleic acid splicing, comprising: an engineered U7 small nuclear RNA (snRNA) comprising a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A).

2. The system of claim 1 , wherein the engineered U7 snRNA further comprises an exonic splicing silencer (ESS) nucleic acid sequence.

3. The system of claim 1, wherein the engineered U7 snRNA does not comprise an ESS nucleic acid sequence.

4. A system for modifying nucleic acid splicing, comprising: an exonic splicing silencer (ESS) nucleic acid sequence; and a target nucleic acid sequence that targets an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A).

5. The system of any one of the preceding claims, wherein the alternatively spliced region is out of frame with an exon of a productive transcript.

6. The system of claim 1 or claim 4, wherein the alternatively spliced region comprises a stop codon, or wherein inclusion of the alternatively spliced region results in a premature stop codon of a mature SCN2A mRNA.

7. The system of claim 1 or claim 4, wherein the SCN2A comprises a human SCN2A.

8. The system of claim 1 or claim 4, wherein the alternatively spliced region is an alternatively spliced exon.

9. The system of claim 1 or claim 4, wherein the alternatively spliced exon comprises exon 13N.

10. The system of claim 9, wherein the exon 13N comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.

11. The system of claim 4, wherein the ESS recruits a protein factor or group of factors that reduce or silence splicing of the RNA encoding SCN2A.

12. The system of claim 1 or claim 4, wherein the target nucleic acid sequence is 10-60 nucleotides in length.

13. The system of claim 1 or claim 4, wherein the target nucleic acid sequence binds to the alternatively spliced region.

14. The system of claim 1 or claim 4, wherein the target nucleic acid sequence is fully reverse complementary or partially reverse complementary to a portion of the alternatively spliced region.Attorney Docket No. 062692-509001 WO15. The system of claim 1 or claim 4, wherein the target nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reverse complementary to a portion of the alternatively spliced region.

16. The system of claim 1 or claim 4, wherein the portion of the alternatively spliced region is within a 5’ half or 5’ end of the alternatively spliced region.

17. The system of claim 1 or claim 4, wherein the portion of the alternatively spliced region comprises nucleotide positions within 50 bp of a 5’ or 3’ end of the alternatively spliced region.

18. The system of claim 4, wherein the target nucleic acid sequence is 3’ or downstream relative to the ESS nucleic acid sequence.

19. The system of claim 1 or claim 4, further comprising a Sm binding site.

20. The system of claim 19, wherein the Sm binding site is 3’ or downstream relative to the ESS nucleic acid sequence or target nucleic acid sequence.

21. The system of claim 1 or claim 4, further comprising a hairpin sequence.

22. The system of claim 21, wherein the hairpin sequence comprises a U7 small nuclearRNA (snRNA) hairpin sequence.

23. The system of claim 21, wherein the hairpin sequence comprises a 3’ hairpin sequence.

24. The system of claim 21, wherein the hairpin sequence is 3’ or downstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, or a Sm binding site.

25. The system of claim 1 or claim 4, wherein the system comprises one or more RNA molecules.

26. The system of claim 1 or claim 4, wherein the RNA is a modified U7 snRNA.

27. The system of claim 1 or claim 4, wherein the system comprises a deoxyribonucleic acid (DNA).

28. The system of claim 1 or claim 4, wherein the system comprises an expression cassette.

29. The system of claim 1 or claim 4, further comprising a promoter sequence.

30. The system of claim 29, wherein the promoter sequence comprises a mouse or human promoter sequence.

31. The system of claim 29, wherein the promoter sequence is 5’ or upstream relative to the ESS nucleic acid sequence, the target nucleic acid sequence, a Sm binding site, or a hairpin sequence.

32. The system of claim 1 or claims 4, further comprising a terminator sequence.

33. The system of claim 32, further wherein the terminator sequence comprises mouse or human terminator sequence.Attorney Docket No. 062692-509001 WO34. The system of claim 32, wherein the terminator sequence comprises a 3’ terminator sequence.

35. The system of claim 32, wherein the terminator sequence is 3’ or downstream relative to a promoter sequence, the ESS nucleic acid sequence, the target nucleic acid sequence, a Sm binding site, or a hairpin sequence.

36. The system of claim 1 or claim 4, wherein the components are combined together within a single nucleic acid.

37. The system of claiml or claim 4, wherein the components are separated among multiple nucleic acids.

38. A pharmaceutical composition comprising the system of claim 1 or claim 4, and a pharmaceutically acceptable carrier.

39. A virus comprising the system of claim 1 or claims 4.

40. The virus of claim 39, comprising a parvovirus.

41. The virus of claim 39, comprising an adeno-associated virus (AAV).

42. A cell comprising the system of claim 1 or claims 4.

43. The cell of claim 42, comprising a neural cell.

44. A method, comprising administering a pharmaceutical composition or a virus comprising the system of claim 1 or claim 4 to a subject.

45. A method of modifying splicing, comprising contacting a pre-mRNA encoding SCN2A with a recombinant U7 small nuclear RNA (snRNA) sequence that induces exclusion of exon 13N from a mature mRNA generated by the pre-mRNA.

46. The method of claim 45, wherein the pre-mRNA is in a cell.

47. The method of claim 46, wherein the cell is in a subject.

48. The method of claim 47, wherein the subject has or is at risk of having a genetic disorder.

49. The method of claim 47, wherein the subject has or is at risk of having a neurodevelopmental disorder.

50. The method of claim 47, comprising administering to the subject a composition that silences or reduces said splicing, the composition comprising the system, virus, or composition of claim 1 or claim 4.

51. A method of treating or preventing a neurodevelopmental disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant U7 small nuclear RNA (snRNA) composition that silences or reduces splicing of an alternatively spliced region of a ribonucleic acid (RNA) encoding sodium channel protein type 2 subunit alpha (SCN2A).

52. The method of claim 51 , wherein the neurodevelopmental disorder comprises intellectual disability, autistic features, or epilepsy.Attorney Docket No. 062692-509001 WO53. The method of claim 51, wherein the alternatively spliced region comprises exon 13N of the RNA.

54. The method of claim 51, wherein the composition comprises the system, virus, or composition of claim 1 or claim 4.

55. The method of claim 45 or claim 51, wherein the contact or administration increases an amount of a productive isoform of SCN2A in the subject, relative to a control or baseline amount of said productive isoform.

56. The method of claim 45 or claim 51, wherein the contact or administration decreases an amount of a non-productive isoform of SCN2A in the subject, relative to a control or baseline amount of said non-productive isoform.

57. The method of claim 51, wherein the administration increases an amount of a NaV1.2 channel or protein (e.g., SCN2A protein) in the subject, relative to a control or baseline amount of the NaV1.2 channel or protein.

58. The method of claim 51, wherein the administration improves sodium transport in the subject, relative to a control or baseline amount.

59. The method of claim 51, wherein the administration prevents the subject from having seizures, or reduces an amount or severity of the seizures of the subject relative to a baseline amount or severity.

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