Intein-mediated functional reconstitution of voltage-gated sodium channels

Abstract

We describe an artificial expression construct for rescuing voltage-gated sodium channel function by intein-mediated reconstitution of voltage-gated sodium channel alpha subunit 1 (Nav1.1), a protein encoded by SCN1A. Rescued voltage-gated sodium channel function can be utilized to treat disorders such as epilepsy, and more specifically, Dravet syndrome.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 482,741, filed February 1, 2023, the contents of which are incorporated herein by reference in their entirety as if set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. MH120095 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0003] Sequence Listing Reference The Sequence Listing accompanying this application is provided in XML format rather than in hard copy, and is incorporated herein by reference. The name of the file containing this Sequence Listing is 44778639_xml. This file is 179,913 bytes in size, was created on January 19, 2024, and was submitted electronically via the Patent Center.

[0004] This disclosure describes the rescue of voltage-gated sodium channel function by intein-mediated reconstitution of voltage-gated sodium channel alpha subunit 1 (Nav1.1), a protein encoded by SCN1A. Rescued voltage-gated sodium channel function can be used to treat disorders such as epilepsy, and more specifically, Dravet Syndrome. [Background technology]

[0005] There are many neurological disorders that require prompt treatment. One such disorder is caused by mutations in SCN1A, a gene that encodes the alpha subunit of the voltage-gated sodium channel (Nav1.1). For example, SCN1A mutations can cause disorders such as Dravet syndrome, myoclonic seizures, and intractable childhood epilepsy with generalized tonic-clonic seizures.

[0006] Epilepsy is a neurological disorder caused by the brain exhibiting a persistent predisposition to two or more epileptic seizures. An epileptic seizure is a transient disturbance of brain function due to abnormally excessive or abnormally synchronized neural activity. Epileptic seizures may manifest as abnormal behavior, abnormal sensations, and sometimes loss of consciousness.

[0007] In particular, Dravet syndrome is a rare, catastrophic, intractable form of epilepsy that begins in infancy. Patients experience prolonged seizures early in the disease. During the second year of life, other types of seizures begin to appear, usually accompanied by developmental delays, resulting in poor development of language and motor skills.

[0008] Children with Dravet syndrome may experience multiple seizures per day. Seizures are highly lethal in Dravet syndrome patients, with 10-16% of diagnosed patients dying during childhood, particularly between the ages of 2 and 4. Additionally, Dravet syndrome patients are at risk for numerous associated conditions, including problems with limb development, poor growth, abnormalities in sleep and circadian rhythms, and chronic infections.

[0009] Of particular concern, children with Dravet syndrome are particularly susceptible to status epilepticus, a condition characterized by a seizure lasting more than five minutes or repeated seizures lasting five minutes without recovery of consciousness in between. This severe condition is classified as an emergency requiring immediate medical intervention and typically involves hospitalization. Persistent convulsive status epilepticus, lasting more than 30 minutes, can be life-threatening and can cause severe brain damage. Frequent hospitalizations for children with Dravet syndrome place a burden on the patients themselves, their families, and caregivers. Furthermore, children with Dravet syndrome require constant supervision, and many patients require institutionalization, increasing the cost of treating Dravet syndrome.

[0010] Currently, various anticonvulsant treatments are available to reduce the occurrence of seizures in individuals with Dravet syndrome; however, such treatments generally produce poor results, resulting in only partial seizure control in the majority of patients. Many anticonvulsant medications, such as clobazam and clonazepam, have undesirable side effects, particularly acute side effects in pediatric patients. Furthermore, certain anticonvulsant medications, particularly sodium channel blockers, can worsen seizures in individuals with Dravet syndrome.

[0011] Cell-type- or cell-class-specific gene delivery using nonpathogenic viral delivery has increasingly shown promise for the treatment of various diseases. Introducing gene regulatory elements, such as specific promoters and enhancers, into the delivery vector has been shown to offer the advantage of conferring specificity to gene expression in specific target cell types. For example, Dimidschstein et al. (Nat Neurosci. 19(12):1743-1749, 2016) developed a gene construct for viral delivery based on adeno-associated virus (AAV) and successfully achieved selective gene expression in gamma-aminobutyric acid (GABAergic) interneurons in the telencephalon, a cell type important for the treatment of epilepsy.

[0012] One of the major drawbacks of using AAV as a selective gene delivery system is the extremely limited size that can be packaged into AAV, especially the large SCN1A gene (6 kb) which exceeds the packaging capacity of AAV (4.7 kb DNA). Summary of the Invention [Means for solving the problem]

[0013] The present disclosure unexpectedly provides expression constructs that enable rapid and high-level protein expression in targeted central nervous system cell types for the purpose of rescuing voltage-gated sodium channel function. The expression constructs of the present disclosure can be used to reverse or alleviate the effects of voltage-gated sodium channel dysfunction caused by damaging SCN1A variants in targeted cell types. SCN1A is typically difficult to deliver due to its large size, which exceeds the packaging capacity of adeno-associated viruses (AAVs). Therefore, as described herein, the coding sequence for SCN1A is packaged into two or more artificial expression constructs, each containing a sequence encoding an N-terminal fragment of a split intein and a sequence encoding a C-terminal fragment of the split intein. The expression products of these two or more artificial expression constructs are spliced ​​together to excise the intein fragments and produce the full-length SCN1A protein.

[0014] In certain embodiments, the present disclosure provides systems and methods for expressing an SCN1A coding sequence in a subject requiring expression of the SCN1A coding sequence. In certain embodiments, the system of the present disclosure includes at least a first artificial expression construct and a second artificial expression construct; the first artificial expression construct includes a first portion of the SCN1A coding sequence, a coding sequence for an N-intein, and a first promoter sequence, the N-intein coding sequence being located at the 3' end of the first portion of the SCN1A coding sequence; and the second artificial expression construct includes a second portion of the SCN1A coding sequence, a coding sequence for a C-intein, and a second promoter sequence, the C-intein coding sequence being located at the 5' end of the second portion of the SCN1A coding sequence. In certain embodiments, when the first artificial expression construct and the second artificial expression construct are expressed in a cell, a protein product of the SCN1A coding sequence is produced by protein splicing. In certain embodiments, the first artificial expression construct and / or the second artificial expression construct comprises an enhancer that directs targeted expression of the first portion and / or the second portion of the coding sequence of SCN1A in a targeted central nervous system cell population.

[0015] In certain embodiments, the present disclosure provides treatment for SCN1A-related disorders, such as Dravet syndrome, myoclonic seizures, and refractory childhood epilepsy with generalized tonic-clonic seizures. For example, as disclosed herein, by administering the artificial expression constructs of the present disclosure, therapeutic efficacy for treating heat-induced seizures, myoclonic seizures, and generalized tonic-clonic seizures can be obtained in well-established in vivo mouse models of these diseases.

[0016] In certain embodiments, the treatment comprises expressing the coding sequence of SCN1A in a subject in need thereof by administering at least a first artificial expression construct and a second artificial expression construct, each of which comprises a portion of the coding sequence of SCN1A, a coding sequence for a split intein, and a promoter sequence.

[0017] In certain embodiments, a split intein is used, including consensus fast intein (Cfa)-N and Cfa-C.

[0018] In certain embodiments, the first portion of the coding sequence for SCN1A and the second portion of the coding sequence for SCN1A, when expressed together and spliced, form a mature protein that rescues the function of a cellular voltage-gated sodium channel. The artificial expression constructs of the present disclosure may further comprise other regulatory elements, as needed or beneficial.

[0019] In certain embodiments, an artificial expression construct of the present disclosure is expressed in every neuron (e.g., after being transduced). In certain embodiments, an artificial expression construct of the present disclosure comprises an hSyn1 promoter and is expressed in a neuron (e.g., after being transduced). In certain embodiments, an artificial expression construct of the present disclosure is expressed in every cell population. In certain embodiments, an artificial expression construct of the present disclosure comprises a CMV promoter and is expressed in a cell population.

[0020] In certain embodiments, enhancers are used to induce gene expression in targeted central nervous system cell populations. In certain embodiments of the artificial expression constructs of the present disclosure, the following enhancers are used to induce gene expression in targeted central nervous system cell populations. The enhancer and target cell population combinations used in this disclosure are listed below, in enhancer / target cell population order: DLX2.0 / forebrain GABAergic neurons; hSyn1 and 4x2C or 8x2C miR binding sites / whole GABAergic neurons; and eHGT_078h / forebrain glutamatergic neurons. In certain embodiments, the artificial expression construct may comprise a truncated promoter or a minimal promoter. In certain embodiments, the truncated promoter comprises a truncated hSyn1 promoter. In certain embodiments, the minimal promoter comprises minB globin.

[0021] In certain embodiments, artificial expression constructs are provided that comprise the features of vector pairs described herein, including a pair of CN3252 and CN3254 vectors, a pair of CN3683 and CN3684 vectors, a pair of CN3251 and CN3253 vectors, a pair of CN3677 and CN3678 vectors, a pair of CN4541 and CN4542 vectors, a pair of CN4217 and CN4218 vectors, or a pair of CN4642 and CN4643 vectors. [Brief explanation of the drawings]

[0022] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby include color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0023] [Figures 1A-1D]We demonstrate efficient intein-mediated reconstitution of human SCN1A from two fragments. (Figure 1A) The design of the bisected NaV1.1 fragments with intein fusion protein tags (Cfa-N and Cfa-C) is shown. The full-length human SCN1A (hSCN1A) encoding NaV1.1 (1998 amino acids long) can be split into two fragments (amino acids 1–1049 at the N-terminus and amino acids 1050–1998 at the C-terminus) and packaged into AAV particles. The cleavage site is at an endogenous cysteine ​​residue (Cys1050), allowing for traceless protein binding. Abbreviations: Cfa: Consensus Fast intein (Stevens et al., J Am Chem Soc. 138(7):2162–5, 2016). (Figure 1B) Plasmid vectors for testing the fusion proteins in cell lines are shown. The N-terminal fragment contains an HA tag at its N-terminus, and the C-terminal fragment contains a FLAG tag at its C-terminus. Furthermore, SYFP2 and mScarlet reporters are added to monitor transfection. Abbreviations: CMV: cytomegalovirus immediate-early / early promoter; IRES2: internal ribosome entry site 2. (Figure 1C) Western blot analysis demonstrates efficient ligation of the two fragments in HEK-293 cells. HEK-293 cells were transfected with the indicated plasmid vectors using polyethyleneimine, and 72 hours later, protein lysates were prepared and analyzed by Western blot analysis. Note that full-length hSCN1A migrated at the expected size of 250 kDa, while the N- and C-terminal fragments migrated at the expected sizes of 134 and 115 kDa, respectively. Although the anti-FLAG blot in lanes 5 and 6, indicated by asterisks, was unexpectedly smeared, the expected protein size was confirmed (all bands of the expected size are indicated by arrows) (Figure 1D). Electrophysiological measurements demonstrated that the split SCN1A channel exhibited functional voltage-gated sodium channel activity in CHO cells.After transfection of CHO cells with the indicated plasmid vectors using polyethyleneimine, the cells were patch-clamped in voltage-clamp mode and currents were measured under stepwise depolarization conditions. The maximum sodium current (Ipeak) was normalized to the cell capacitance, which is dependent on cell size. High currents were obtained with both full-length hSCN1A and intein-fused hSCN1A, whereas currents were low under control conditions.

[0024] [Figures 2A-2F]We demonstrate cell type-specific delivery of AAV-assembled hSCN1A in the mouse brain. (Figure 2A) The design of the AAV vector system for SCN1A delivery is shown, using two bisected SCN1A fragments with intein fusion protein tags, the inhibitory neuron-specific enhancer DLX2.0, and a minimal promoter (minP) driving each fragment. Abbreviations: WPRE3: Woodchuck Hepatitis Virus Post-Translational Regulatory Element 3; pA: Poly(A) site; LITR: Left Intermediate Transverse Receptor (ITR); RITR: Right Intermediate Transverse Receptor (ITR). (Figure 2B) We demonstrate intein-mediated production of full-length hSCN1A from AAV vectors in the mouse brain. The indicated AAVs were packaged in PHP.eB and delivered intravenously to mice (intravenous delivery of 3 x 10 genome copies [GC] of each AAV vector into adult mice). After 3 weeks, brains were removed, and plasma membrane protein lysates were prepared from the primary visual cortex and Western blots were performed. Only when the N-terminal and C-terminal fragment vectors were delivered together did full-length SCN1A (NaV1.1) protein be produced. The vectors containing only one of the fragments were not detectable, likely due to insufficient enrichment from plasma membrane protein lysates without purification of the unbound fragments, or the short half-life of the protein. (Figure 2C) Specific detection of each hSCN1A fragment in mouse brain by immunohistochemistry (IHC) is shown. The indicated AAV / PHP.eB vectors were administered to neonatal mice via the intracerebroventricular route (ICV, 3 x 1010 GC each). Three weeks later, brains were removed, and the expression pattern of each fragment was assessed by immunohistochemistry (shown here in the primary visual cortex (VISp)). Each fragment could be detected alone or together and was found in neuronal cell bodies and processes of scattered neurons (putative inhibitory neurons). (Figure 2D) Expression of assembled hSCN1A was observed in scattered neurons (putative interneurons) throughout the forebrain. N-terminal and C-terminal vectors were delivered to adult mice by ICV injection (3 × 10 10 GC each), and expression was analyzed by IHC.(Figure 2E) Co-staining of assembled hSCN1A with Gad67, an interneuron marker, suggested that numerous interneurons in the VISp expressed assembled hSCN1A. The same mouse is shown in Figure 2D. (Figure 2F) Quantitative analysis of the biodistribution of VISp Gad67+ neurons revealed high specificity of expression in inhibitory neurons (defined as the percentage of marker+ cells that also express Gad67+; generally 95–100% across cortical layers), suggesting that numerous VISp Gad67+ neurons express HA and FLAG markers. Complete labeling (defined as the percentage of marker+ / Gad67+ cells relative to total Gad67+ cells) was dose-dependent, with more expressed neurons at a dose of 3×1010 GC than at a dose of 1×1010 GC, and higher expression levels were observed with highly purified AAV vector preparations produced by Packgene than those produced by the Allen Institute viral core. For reference, mouse K9263 is shown in Figures 2D and 2E.

[0025] [Figure 3] In vivo testing of hSCN1A split intein AAV in Dravet syndrome mice is shown. The diagram shows the experimental setup for testing the function of the DLX2.0-hSCN1A split intein vector. The first and second halves of the DLX2.0 split intein SCN1A fusion vector were packaged into serotype PHP.eB AAV vectors and injected into Dravet syndrome mutant or control animals at low doses (1 x 10 GC per vector) or high doses (3 x 10 GC per vector), or uninjected Dravet syndrome mutant or control animals. In the high-dose group, vectors obtained from two sources were tested: in-house packaged virus and virus obtained from a commercial vendor (PackGene). Neonatal mice were injected with virus between P0 and P3. Survival was assessed by monitoring until postnatal day 70 (P70).

[0026] [Figure 4] The DLX2.0-SCN1A split intein vector completely rescues mortality in the Dravet syndrome model. Kaplan-Meier curves showed that both low- and high-dose regimens rescued mortality in Dravet syndrome mice, and that the virus obtained from Packgene completely rescued early mortality.

[0027] [Figures 5A-5C] The DLX2.0 split intein SCN1A vector significantly rescued heat-induced seizures (Figure 5A, Figure 5B). Both in-house (IH)-produced and commercially available (PG)-produced viruses demonstrated sufficient resistance to heat-induced myoclonic seizures, shifting the body temperature at which myoclonic seizures occurred by approximately 2°C (Figure 5A) and significantly reducing seizure burden (cumulative number of seizures) (Figure 5B). (Figure 5C) Both in-house (IH)-produced and commercially available (PG)-produced viruses demonstrated nearly complete rescue of heat-induced generalized tonic-clonic seizures.

[0028] [Figures 6A-6B] The hSyn1-hSCN1A split intein vector, unlike the DLX2.0-hSCN1A split intein vector, was toxic in young mice (Figure 6A). Neonatal injection of either a high (3 × 10) or low (1 × 10) dose of the hSyn1-hSCN1A split intein vector resulted in lethality, whereas no lethality was observed with the DLX2.0-hSCN1A split intein vector. This result suggests that early lethality is due to neuronal-wide expression driven by the hSyn1 promoter (Figure 6B). Both high- and low-dose hSyn1-hSCN1A vectors continued to cause death in some Dravet syndrome mutant mice after weaning at 3 weeks of age. The low- and high-dose vectors were compared with uninjected controls or Dravet syndrome mutant mice injected with the PHP.eB-CN1390:DLX2.0-SYFP2 control vector.

[0029] [Figures 7A-7B] High doses of the hSyn1-hSCN1A split intein vector, but not low doses, can rescue heat-induced seizures. Neonatal Dravet syndrome model mice were treated with various hSCN1A split intein vectors or without treatment. Dravet syndrome mice surviving beyond P70 were subjected to 42°C heat induction and monitored for myoclonic seizures (Figure 7A) or generalized tonic-clonic seizures (Figure 7B). Note that high doses of DLX2.0-hSCN1A significantly reduced myoclonic and generalized tonic-clonic seizures without toxicity to young mice (Figure 6A, Figure 6B). High doses of whole-neuron hSyn1-hSCN1A split intein reduced seizures but caused significant toxicity (Figure 6A, Figure 6B). In contrast, a low dose of whole-neuron hSyn1-hSCN1A split intein did not reduce seizures compared with untreated mice.

[0030] [Figures 8A-8D]High-dose DLX2.0-hSCN1A split intein vector completely rescued death and seizures in the Dlx5 / 6-Cre;Scn1afl / + Dravet model (Figure 8A). A schematic diagram of the Dlx5 / 6-Cre;Scn1afl / + model mating is shown. Experiments were performed using offspring obtained through this mating scheme. In this model, one copy of Scn1a is deleted exclusively in GABAergic cells in the forebrain. These GABAergic cells are the same cell type whose expression is induced by DLX2.0 (Figure 8B). Kaplan-Meier curves showed that all untreated Dlx5 / 6-Cre;Scn1afl / + mutant mice died by the fourth week of age. In contrast, none of the mice injected with the DLX2.0-hSCN1A split intein vector died after the 10th week of age. (Fig. 8C) When mice were tested for heat-induced seizures after P70, none of the mice (n = 7) exhibited myoclonic seizures. (Fig. 8D) When mice were tested for heat-induced seizures after P70, none of the mice (n = 7) exhibited generalized tonic-clonic seizures.

[0031] [Figure 9] Alternative sites for splitting human SCN1A to halve the size of AAV are shown. (Figure 9A) A split protein design with a cutpoint at Cys1050 is shown. This design was used in Figures 1-8. Note that the endogenous cysteine ​​residue is required for traceless ligation of the fragments to reconstitute the full-length protein without mutations. (Figures 9B and 9C) Alternative split intein designs with cutpoints at Cys957 (Figure 9B) or Cys948 (Figure 9C) are shown. These cutpoints result in better AAV size and packaging efficiency of the N-terminal fragment than hSCN1A-CO-Nterm1049, which uses Cys1050 as the cutpoint, resulting in better expression. However, these designs place the intein junction in the extracellular / luminal space.

[0032] [Figure 10]Sequences supporting this disclosure are set forth below: 1xhI56i(core) (core of human I56i enhancer) (SEQ ID NO: 1), DLX2.0 (3x human I56i core) (SEQ ID NO: 2), eHGT_078h (SEQ ID NO: 55), hSynl promoter (SEQ ID NO: 52), CMV promoter (SEQ ID NO: 53), truncated hSynl promoter (SEQ ID NO: 54), 4x2C (SEQ ID NO: 56), 8x2C (SEQ ID NO: 87), Cfa-N (SEQ ID NO: 57), Cfa-C (SEQ ID NO: 58), hSCN1A-CO-Nterm1049-intron (SEQ ID NO: 59), hSCN1A-CO-Cterm949-intron Intron (SEQ ID NO: 60), hSCN1A-CO-Nterm956-intron (SEQ ID NO: 61), hSCN1A-CO-Cterm1042-intron (SEQ ID NO: 62), hSCN1A-CO-Nterm947-intron (SEQ ID NO: 63), hSCN1A-CO-Cterm1051-intron (SEQ ID NO: 64), human SCN1A (SEQ ID NO: 65), β-globin minimal promoter (pBGmin / minBGlobin / minBGprom) (SEQ ID NO: 3), minCMV promoter (SEQ ID NO: 4), mutant minCMV promoter (SacIRE site removed) (SEQ ID NO: 5), minRho promoter (SEQ ID NO: 6), minRho* promoter (SEQ ID NO: 7), Hsp68 minimal promoter (proHsp68) (SEQ ID NO: 8), SYFP2 (SEQ ID NO: 9), EGFP (SEQ ID NO: 10), mScarlet (SEQ ID NO: 66), 3xNLS (SEQ ID NO: 67), optimized Flp recombinase (FlpO) (SEQ ID NO: 11), improved Cre recombinase (iCre) (SEQ ID NO: 12), SP10 insulator (SP10ins) (SEQ ID NO: 13), 3xSP10ins (SEQ ID NO: 14), 3XFLAG version 1 (SEQ ID NO: 68), 3XFLAG version 2 (SEQ ID NO: 15), 2xHA (SEQ ID NO: 69) , 10aa (SEQ ID NO: 16), H2B (SEQ ID NO: 17), WPRE3 (SEQ ID NO: 18), WPRE (SEQ ID NO: 19), BGHpA (SEQ ID NO: 20), hGHpA (SEQ ID NO: 21), SV40pA (SEQ ID NO: 70), ShortPolyA (SEQ ID NO: 71), IRES2 (SEQ ID NO: 72), P2A (SEQ ID NO: 22), T2A (SEQ ID NO: 23), E2A (SEQ ID NO: 24), F2A (SEQ ID NO: 25), exemplary plasmid backbone 1 - left ITR (SEQ ID NO: 26), exemplary plasmid backbone 1 - right ITR (SEQ ID NO: 27), exemplary plasmid backbone 2 - left ITR (SEQ ID NO: 28), exemplary plasmid backbone 2 - right ITR (SEQ ID NO: 29), PHP.eB capsid (SEQ ID NO: 30), AAV9 VP1 capsid protein (SEQ ID NO:31), tet-transactivator version 2 (tTA2) (SEQ ID NO:32), GTPase HRas [Homo sapiens] (SEQ ID NO:33), CN3252 (SEQ ID NO:73), CN3254 (SEQ ID NO:74), CN3683 (SEQ ID NO:75), CN3684 (SEQ ID NO:76), CN3251 (SEQ ID NO:77), CN3253 (SEQ ID NO:78), CN3677 (SEQ ID NO:79), CN3678 (SEQ ID NO:80), CN4541 (SEQ ID NO:81), CN4542 (SEQ ID NO:82), CN4217 (SEQ ID NO:83), CN4218 (SEQ ID NO:84), CN4642 (SEQ ID NO:85), and CN4643 (SEQ ID NO:86). DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure unexpectedly provides expression constructs that enable rapid and high-level protein expression in targeted central nervous system cell types for the purpose of rescuing voltage-gated sodium channel function. The expression constructs of the present disclosure can be used to reverse or alleviate the effects of voltage-gated sodium channel dysfunction caused by damaging SCN1A variants in targeted cell types. SCN1A is typically difficult to deliver due to its large size, which exceeds the packaging capacity of adeno-associated viruses (AAVs). Therefore, as described herein, the coding sequence for SCN1A is packaged into two or more artificial expression constructs, each containing a sequence encoding an N-terminal fragment of a split intein and a sequence encoding a C-terminal fragment of the split intein. The expression products of these two or more artificial expression constructs are then spliced ​​together to produce the encoded full-length SCN1A protein and the split intein.

[0034] In certain embodiments, the present disclosure provides systems and methods for expressing an SCN1A coding sequence in a subject requiring expression of the SCN1A coding sequence. In certain embodiments, the disclosed system includes at least a first artificial expression construct and a second artificial expression construct; the first artificial expression construct includes a first portion of the SCN1A coding sequence, a coding sequence for an N-intein, and a first promoter sequence, the N-intein coding sequence being located at the 3' end of the first portion of the SCN1A coding sequence; and the second artificial expression construct includes a second portion of the SCN1A coding sequence, a coding sequence for a C-intein, and a second promoter sequence, the C-intein coding sequence being located at the 5' end of the second portion of the SCN1A coding sequence. In certain embodiments, when the first artificial expression construct and the second artificial expression construct are expressed in a cell, a protein product of the SCN1A coding sequence is produced by protein splicing. In certain embodiments, the first artificial expression construct and / or the second artificial expression construct comprises an enhancer that directs targeted expression of the first portion and / or the second portion of the coding sequence of SCN1A in a targeted type of central nervous system cell.

[0035] In certain embodiments, the present disclosure provides treatment for SCN1A-related disorders, such as Dravet syndrome, myoclonic seizures, and refractory childhood epilepsy with generalized tonic-clonic seizures. For example, as disclosed herein, by administering the artificial expression constructs of the present disclosure, therapeutic efficacy for treating febrile seizures, myoclonic seizures, and generalized tonic-clonic seizures can be obtained in well-established in vivo mouse models of these diseases.

[0036] In certain embodiments, the treatment comprises expressing the coding sequence of SCN1A in a subject in need thereof by administering at least a first artificial expression construct and a second artificial expression construct, each of which comprises a portion of the coding sequence of SCN1A, a coding sequence for a split intein, and a promoter sequence.

[0037] In certain embodiments, a split intein containing Cfa-N and a split intein containing Cfa-C are used. In certain embodiments, the coding sequence for the Cfa-N intein comprises SEQ ID NO: 57. In certain embodiments, the coding sequence for the Cfa-C intein comprises SEQ ID NO: 58.

[0038] In certain embodiments, the first portion of the coding sequence for SCN1A and the second portion of the coding sequence for SCN1A, when expressed together and spliced, form a mature protein that rescues the function of a cellular voltage-gated sodium channel. The artificial expression constructs of the present disclosure may further comprise other regulatory elements, as needed or beneficial.

[0039] In certain embodiments, the artificial expression constructs of the present disclosure are expressed in any neuron. In certain embodiments, the artificial expression constructs of the present disclosure comprise an hSyn1 promoter and are expressed in neurons. In certain embodiments, the artificial expression constructs of the present disclosure are expressed in any cell line. In certain embodiments, the artificial expression constructs of the present disclosure comprise a CMV promoter and are expressed in cell lines.

[0040] In certain embodiments, enhancers are used to induce gene expression in targeted central nervous system cell populations. In certain embodiments of the artificial expression constructs of the present disclosure, the following enhancers are used to induce gene expression in targeted central nervous system cell populations. The enhancer and target cell population combinations used in this disclosure are listed below, in enhancer / target cell population order: DLX2.0 / forebrain GABAergic neurons; hSyn1 and 4x2C or 8x2C miR binding sites / whole GABAergic neurons; and eHGT_078h / forebrain glutamatergic neurons. In certain embodiments, the artificial expression construct may comprise a truncated promoter or a minimal promoter. In certain embodiments, the truncated promoter comprises a truncated hSyn1 promoter. In certain embodiments, the minimal promoter comprises minB globin.

[0041] In certain embodiments, pairs of artificial expression constructs are provided that comprise the features of vectors described herein, including a pair of CN3252 and CN3254 vectors, a pair of CN3683 and CN3684 vectors, a pair of CN3251 and CN3253 vectors, a pair of CN3677 and CN3678 vectors, a pair of CN4541 and CN4542 vectors, a pair of CN4217 and CN4218 vectors, or a pair of CN4642 and CN4643 vectors.

[0042] Various aspects of the present disclosure are described in more detail below, along with additional options. The various aspects of the present disclosure are described under the following headings: (i) Artificial Expression Constructs and Vectors for Targeted Expression of Genes in Target Cell Types; (ii) Compositions for Administration; (iii) Cell Lines Containing Artificial Expression Constructs; (iv) Transgenic Animals; (v) Methods of Use; (vi) Kits and Commercial Packages; (vii) Exemplary Embodiments; and (viii) Conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the present disclosure.

[0043] (i) Artificial expression constructs and vectors for targeted expression of genes in target cell types The systems and methods disclosed herein include expressing an SCN1A coding sequence in a subject in need thereof by administering at least a first artificial expression construct and a second artificial expression construct. In certain embodiments, the first artificial expression construct comprises a first portion of the SCN1A coding sequence, a coding sequence for an N-intein, and a first promoter sequence; the N-intein coding sequence is located at the 3' end of the first portion of the SCN1A coding sequence; and the second artificial expression construct comprises a second portion of the SCN1A coding sequence, a coding sequence for a C-intein, and a second promoter sequence, the coding sequence for the C-intein being located at the 5' end of the second portion of the SCN1A coding sequence. In certain embodiments, when the first and second artificial expression constructs are expressed in a cell, a protein product of the SCN1A coding sequence is produced by protein splicing. In certain embodiments, the N-terminal portion of the coding sequence for SCN1A and the C-terminal portion of the coding sequence for SCN1A, when expressed together and spliced, form a mature protein that rescues the function of a cellular voltage-gated sodium channel. The artificial expression constructs of the present disclosure may further comprise other regulatory elements as needed or beneficial.

[0044] Inteins are a type of autocatalytic enzyme that possess both protease and ligase activities. Inteins are internal protein elements that self-cleave from host proteins and catalyze the ligation of adjacent sequences (exteins) via peptide bonds. Intein excision is a post-translational process that does not require auxiliary enzymes or cofactors. This self-cleavage process is called "protein splicing" because it resembles the splicing of RNA introns from pre-mRNA (Perler F et al., Nucl Acids Res. 22: 1125-1127 (1994)). The internal protein sequence segment is called the "intein," the external protein sequence segment is called the "extein," the segment upstream of the extein is called the "N-extein," and the segment downstream of the extein is called the "C-extein." This protein splicing process results in two stable proteins: the mature protein and the intein.

[0045] Known inteins share low sequence similarity, with conserved residues only at the N- and C-termini. Most intein sequences begin with Ser or Cys and end with His-Asn or His-Gln. The first amino acid of the C-extein is an invariant residue, Ser, Thr, or Cys, whereas the residues of the N-extein preceding the intein are not conserved (Perler F. 2002, Nucl. Acids Res. 30: 383-384). However, recently, residues near the intein splicing junction in the N- and C-terminal exteins have been found to promote or inhibit protein splicing (Amitai G et al. 2009, Proc. Natl. Acad. Sci. USA. 106: 11005-11010).

[0046] One type of intein, called a "split intein," utilizes the selective and extremely tight association of two complementary intein fragments, designated N-intein and C-intein, to form an active intein enzyme (Shah NH, et al, J. Amer. Chem. Soc. 135: 18673-18681; Dassa B., et al, Nucl. Acids Res., 37:2560-2573 (2009)). The two fragments of a split intein are encoded by two genes that are transcribed and translated separately. The so-called split intein self-associates and catalyzes protein trans-splicing activity. Split inteins have been identified in various cyanobacteria and archaea (Caspi et al., Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al., J Mol Biol. 556: 1093-1106 (2006); Dassa B. et al., Biochemistry. 46:322-330 (2007); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H. et al., Proc Natl Acad Sci USA. £5:9226-9231 (1998); and Zettler J. et al., FEBS Letters. 553:909-914 (2009)), but have not yet been found in eukaryotes. Recently, bioinformatics analysis of environmental metagenomic data has uncovered 26 loci with novel genomic organizations. In each locus, the coding region for a conserved enzyme is interrupted by a split intein, with a free-standing endonuclease gene inserted between the intein subdomains. Five of these loci have been fully assembled: DNA helicase (gp41-l, gp41-8); inosine-5'-monophosphate dehydrogenase (IMPDH-1); and ribonucleotide reductase catalytic subunits (NrdA-2 and NrdJ-1).Such fragmented gene organization is primarily found in phages (Dassa et al., Nucleic Acids Research. 57:2560-2573 (2009)).

[0047] Currently, over 350 inteins have been identified, each with a different catalytic rate for the splicing reaction. Intein nomenclature is based on the scientific name of the organism in which they are found. For example, the Ssp intein was first isolated from the genus Synechocystis, while the faster splicing intein, Npu, was first isolated from Nostoc punctiforme. A database containing a partial list of known inteins is available at http: / / www.biocenter.helsinki.fi / bi / iwai / lnBase / tools.neb.com / inbase / list.html.

[0048] In certain embodiments, the intein of the present disclosure comprises a Cfa intein, an Ssp intein, a gp41-1 intein, an IMPDH-1 intein, an Nrdj-1 intein, a gp41-8 intein, or an Npu intein. In certain embodiments, the intein of the present disclosure has a function similar to that of a Cfa intein. In certain embodiments, the intein of the present disclosure comprises a Cfa intein. As used herein, "function similar to that of a Cfa intein" means that an expression construct of the present disclosure contains a variant of a Cfa intein but is still capable of constructing a functional protein (e.g., a voltage-gated sodium channel). In certain embodiments, the intein of the present disclosure comprises an N-intein and a C-intein that form an intein when expressed together and spliced. For example, a Cfa intein comprises Cfa-N and Cfa-C. In certain embodiments, one or more inteins can be used in each artificial expression construct. In certain embodiments, the mature protein can be expressed by splitting the coding sequence into two fragments, inserting the N-terminal portion of the coding sequence and the N-intein into a first artificial expression construct, inserting the C-terminal portion of the coding sequence and the C-intein into a second artificial expression construct, and administering the first and second artificial expression constructs to a cell. In certain embodiments, the mature protein can be expressed by dividing the coding sequence into three fragments, inserting the N-terminal portion of the coding sequence and a first N-intein into a first artificial expression construct, inserting the middle portion of the coding sequence and a first C-intein and a second N-intein into a second artificial expression construct, and inserting the C-terminal portion of the coding sequence and a second C-intein into a second artificial expression construct, such that the first N-intein and the first C-intein specifically splice together to form an intein, and the second N-intein and the second C-intein specifically splice together to form another intein, and administering the first, second, and third artificial expression constructs to a cell.Similarly, by using an appropriate number of inteins, a mature protein can be formed from several fragments. In certain embodiments, the coding sequence for the Cfa-N intein comprises SEQ ID NO: 57. In certain embodiments, the coding sequence for the Cfa-C intein comprises SEQ ID NO: 58.

[0049] In certain embodiments, the artificial expression constructs of the present disclosure comprise regulatory elements that direct gene expression in targeted or non-targeted cell populations. Specific examples of regulatory elements that may be used in the artificial expression constructs disclosed herein include DLX2.0, the minBglobin promoter, the hSyn1 promoter, the CMV promoter, the truncated hSyn1 promoter, the 4x2C miR binding site, the 8x2c miR binding site, and eHGT_078h.

[0050] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the amount of transcription associated with a promoter, can function in either the forward or reverse orientation relative to the promoter and the coding sequence to be transcribed, and can be located upstream or downstream relative to the promoter or the coding sequence to be transcribed. A variety of methods or techniques are known in the art for measuring the function of enhancer element sequences.

[0051] In certain embodiments, the enhancer used in the target type of central nervous system cells is an enhancer that is only utilized in the target type of central nervous system cells, or an enhancer that is primarily utilized in the target type of central nervous system cells.The enhancer used in the target type of central nervous system cells is an enhancer that enhances the expression of genes in the target type of central nervous system.In certain embodiments, the enhancer used in the target type of central nervous system cells enhances the expression of genes in the target type of central nervous system, but does not substantially induce the expression of genes in other non-target cells, and therefore is also a target central nervous system enhancer with cell type-specific transcriptional activity.

[0052] In certain embodiments, the regulatory element may include a neuronal tag (e.g., mAGNET) guided by an miRNA, which tag is referred to herein as an "miR binding site." Exemplary miR binding sites include 4X2C, 4X3C, and 8X2C. In certain embodiments, 4X2C includes the sequence set forth in SEQ ID NO: 56. Additional miR binding sites include 4xMir128_4xMir221_BPL, 4xMir128_4xMir221_4xMir183_4xMir122_BPL, and 4xMir183_4xMir122_BPL.

[0053] When a heterologous coding sequence operably linked to an enhancer disclosed herein is expressed in a cell type that is targeted, the administered heterologous coding sequence is expressed in the intended cell type.

[0054] When a heterologous coding sequence is selectively expressed in selected cells, the administered heterologous coding sequence is expressed in the intended cell type but is not substantially expressed in other cell types. This is described in more detail below. In certain embodiments, not substantially expressed in other cell types means less than 50% expression in the reference cell compared to the target cell; less than 40% expression in the reference cell compared to the target cell; less than 30% expression in the reference cell compared to the target cell; less than 20% expression in the reference cell compared to the target cell; or less than 10% expression in the reference cell compared to the target cell. In certain embodiments, the term "reference cell" refers to a non-target cell. The non-target cell may be present in the same anatomical structure as the target cell and / or may project to a common anatomical region. In certain embodiments, the reference cell is present in an anatomical structure adjacent to the anatomical structure containing the target cell. In certain embodiments, the reference cell is a non-target cell with a gene expression profile different from that of the target cell.

[0055] In certain embodiments, the transcription of the coding sequence may be expressed at a low level in unselected cells, for example, at less than 1%, or 1%, 2%, 3%, 5%, 10%, 15%, or 20% of the transcription level in selected cells. In certain embodiments, the targeted central nervous system cells are the only type of cells that can express the correct combination of transcription factors that can bind to the enhancers disclosed herein and induce gene expression. Thus, in certain embodiments, expression occurs exclusively in the targeted cell type.

[0056] In certain embodiments, target cells (e.g., neuronal and / or non-neuronal cells) can be identified based on transcriptional profiles, such as those described in Tasic et al., Nature 563, 72-78 (2018) and Hodge et al., Nature 573, 61-68 (2019). For reference, various types of cells and their salient features are described below.

[0057] Subclassification of GABAergic neurons in the neocortex: Overall: Expresses the GABA synthesis genes Gad1 / GAD1 and / or Gad2 / GAD2. Lamp5-, Sncg-, Serpinf1-, and Vip-positive GABAergic neurons: These neurons arise from neural progenitor cells derived from the caudal ganglia primordium (CGE) or preoptic area (POA) during development. Sst and Pvalb-positive GABAergic neurons: These neurons arise from neural precursor cells derived from the medial ganglia primordium (MGE) during development. Lamp5-positive GABAergic neurons are found in numerous neocortical layers, especially in the upper layers (L1-L2 / 3), and primarily have neurogliaform and single bouquet cell morphologies. Lamp5_Lhx6-positive GABAergic neurons: a subset of Lamp5-positive GABAergic neurons that co-express Lamp5 and Lhx6. Sncg-positive GABAergic neurons: Found in many neocortical layers, they share common molecules with Lamp5-positive and Vip-positive neurons, but the expression of Lamp5 and vasoactive intestinal peptide (Vip) is inconsistent, whereas the expression of Sncg is consistent. Serpinf1-positive GABAergic neurons: Found in many neocortical layers, they share molecules common to Sncg- and Vip-positive cells, but the expression of Sncg and Vip is inconsistent, whereas the expression of Serpinf1 is consistent. Vip-positive GABAergic neurons: Found in many neocortical layers, but particularly common in the upper layers (L1-L4), they highly express the neurotransmitter Vip. Sst-positive GABAergic neurons: Found in many neocortical layers, but particularly common in the lower layers (L5-L6). They highly express the neurotransmitter somatostatin (Sst) and frequently block dendritic inputs to postsynaptic neurons. This subclass includes sleep-active Sst Chodl neurons (which additionally express Nos1 and Tacr1), which are significantly different from other Sst neurons but express several shared marker genes, including Sst. In humans, SST gene expression is frequently detected in a subtype of LAMP5+ GABAergic neurons in layer 1. Pvalb-positive GABAergic neurons are found in many neocortical layers, but are particularly prevalent in the lower layers (L5-L6). These neurons express high levels of the calcium-binding protein parvalbumin (Pvalb) and the neuropeptide Tac1, and often attenuate the output of postsynaptic neurons. Most fast-firing GABAergic neurons express high levels of Pvalb. This subclass includes chandelier cells, which have a characteristic chandelier-like morphology and express the markers Cpne5 and Vipr2 in mice and NOG and UNC5B in humans. Meis2: A distinct subclass defined by a single cell type, neocortical GABAergic neurons, that express the Meis2 gene but do not express several other genes expressed by other neocortical GABAergic neurons (e.g., Thy1 and Scn2b). These cells are found in L6b and subcortical white matter.

[0058] Subclassification of neocortical glutamatergic neurons: Overall: These neurons express the glutamate transmitters Slc17a6 and / or Slc17a7. Both neurons express Snap25 and lack Gad1 / Gad2 expression. L2 / 3 IT glutamatergic neurons: Predominantly located in layers 2 and 3, with predominantly intratelencephalic (intercortical) projections. L4 IT glutamatergic neurons: Predominantly located in layer 4, with primarily local or intratelencephalic (intercortical) projections. L5 IT glutamatergic neurons: Predominantly located in layer 5, with predominantly intratelencephalic (intercortical) projections. Also called L5a. L5 PT glutamatergic neurons: Predominantly located in layer 5, they primarily project cortico-subcortical (pyramidal or corticofugal) neurons. Also known as L5b, L5 CF (corticofugal), or L5 ET (extratelencephalic). This subclass includes cells in the primary motor cortex and adjacent areas. These cells are corticospinal neurons associated with motor neuron / movement disorders (e.g., ALS). This subclass also includes thick tufted pyramidal neurons, including specialized subtypes found only in certain regions, such as Betz cells, Meynert cells, and von Economo cells. L5 NP glutamatergic neurons: Predominantly located in layer 5, they project primarily to nearby areas. L6 CT glutamatergic neurons: Predominantly located in layer 6, they primarily project to the corticothalamus. L6 IT glutamatergic neurons: Predominantly located in layer 6, with predominantly intratelencephalic (intercortical) projections. L6 IT Car3 glutamatergic neurons: Most densely present in the claustrum and endopyriform nucleus, with sparser distribution throughout L6 in multiple cortical areas, including the primary visual cortex. These neurons primarily project intratelencephalon (intercortical). Additional marker genes for claustrum-enriched neurons include Gnb4 and Ntng2. L6b glutamatergic neurons: Predominantly located in the neocortical subplate (L6b), they project locally (near the cell body), with some also projecting cortically from the VISp to the anterior cingulate bundle and cortico-subcortically to the thalamus. CR neurons: a distinctive subclass defined by a single type found in L1. Cajal-Retzius cells express the characteristic molecular markers Lhx5 and Trp73.

[0059] Classification and subclassification of thalamic GABAergic neurons: Overall: Expresses the GABA synthesis genes Gad1 / GAD1 and / or Gad2 / GAD2. Thalamic reticular nucleus (TRN) neurons express the GABA synthesis genes Gad1 / GAD1 and Pvalb / PVALB.

[0060] Classification and subclassification of thalamic glutamatergic neurons: Whole glutamatergic neurons: express the glutamate transporters Slc17a6 / SLC17A6 and / or Slc17a7 / SLC17A7. Glutamatergic neurons lack Gad1 / Gad2 expression and express one or more of the following marker genes: Synpo2 / SYNPO2, Rgs16 / RGS16, Plekhg1 / PLEKHG1, and Prkcd / PRKCD. Glutamatergic neurons in the parafascicular nucleus (Pf): The parafascicular nucleus (Pf) is the posterior component of the intralaminar thalamic nucleus. It plays a role in the feedback system of the basal ganglia-thalamo-cortical circuit, which is crucial for cognitive processes.

[0061] Target striatal cell types:

[0062] The striatum (Str) is important in converting cortical activity into voluntary movement. In humans / primates, the structures corresponding to the striatum are called the putamen, caudate nucleus, and ventral striatum, with the ventral striatum including the nucleus accumbens. In rodents, the striatum includes the dorsal striatum, which includes the nucleus accumbens. Therefore, the putamen and caudate nucleus in humans / primates together correspond to the dorsal striatum in rodents.

[0063] Classification and subclassification of striatal cells: Medium spiny neurons (total): These neurons account for 95% of striatal neurons and are known to express the GABA synthesis genes Gad1 / GAD1 and Gad2 / GAD2, as well as Ppp1r1b / PPP1R1B. Herein, medium spiny neurons that express Drd3 are referred to as Drd3+ medium spiny neurons. Medium spiny neurons, direct pathway projection type: They account for nearly 50% of striatal neurons and are enriched in Drd1 / DRD1, Pdyn / PDYN, and Slc35d3 / SLC35D3. The main axonal projections from direct pathway medium spiny neurons are to the substantia nigra pars reticulata (SNr) or the globus pallidus interna (GPi). Medium spiny neurons, indirect pathway projection type: These neurons account for nearly 50% of striatal neurons and are enriched for Drd2 / DRD2, Adora2a / ADORA2A, Gpr6 / GPR6, and Penk / PENK. The main axonal projection destination of indirect pathway medium spiny neurons is the external segment of the globus pallidus (GPe). Striatal interneurons - cholinergic: A rare population of interneurons that accounts for 1% of striatal neurons. These local interneurons have large cell bodies, few dendritic spines, express Chat / CHAT, and are known to release the neurotransmitter acetylcholine.

[0064] The cerebellum is located at the posterior part of the cerebrum. It processes inputs from the cerebral motor cortex, various brainstem nuclei, and sensory receptors. Two types of neurons, Purkinje cells and granule cells, play a major role in the cerebellar circuitry. Additionally, the cerebellum receives dopaminergic, serotonergic, noradrenergic, and cholinergic inputs. Cerebellar Purkinje cells: Large GABAergic neurons that are the only projection neurons from the cerebellum. The cell bodies of cerebellar Purkinje cells form a single layer known as the "Purkinje cell layer" and express parvalbumin. Deep cerebellar nucleus neurons: Neurons present in the deep cerebellar nucleus structure. These neurons include glutamatergic and GABAergic cells that express the Pvalb gene. · Molecular layer interneurons (MLIs): intracerebellar neurons that participate in spatially structured networks via chemical and electrical synapses. Chandelier cells: specialized GABAergic interneurons that selectively innervate pyramidal neurons. Striatal medium spiny neurons: These are the main striatal neurons. They receive synaptic input from glutamatergic and dopaminergic afferents.

[0065] Dopaminergic neurons secrete dopamine. Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system. Loss of dopaminergic neurons is associated with Parkinson's disease, one of the most well-known human neurological disorders. Studies on the developmental pathways involved in the development of dopaminergic neurons in the brain have identified several specific transcription factors, including Nurr1, Lmx1b, and Pitx3, all of which have been shown to be important in the development of the midbrain dopaminergic system.

[0066] In certain embodiments, the coding sequence is a heterologous coding sequence encoding an effector element. The effector element is a sequence that is expressed to achieve a desired effect, and the effector element actually achieves the desired effect. Examples of effector elements include reporter genes / proteins and functional genes / proteins. In certain embodiments, the effector element is a protein that rescues the function of voltage-gated sodium channels. In certain embodiments, the coding sequence is SCN1A.

[0067] In certain embodiments, the artificial expression constructs of the present disclosure can deliver SCN1A as several SCN1A fragments delivered by several artificial expression constructs. For example, SCN1A can be delivered in the form of a first artificial expression construct comprising a first portion of the SCN1A coding sequence and a second artificial expression construct comprising a second portion of the SCN1A coding sequence. In certain embodiments, the first portion of the SCN1A coding sequence is an N-terminal portion of the SCN1A coding sequence, and the second portion of the SCN1A coding sequence is a C-terminal portion of the SCN1A coding sequence. The SCN1A coding sequence can be split into an N-terminal portion and a C-terminal portion at any break point that results in expression of a functional SCN1A molecule via intein fusion. In certain embodiments, the N-terminal portion of the coding sequence for SCN1A comprises hSCN1A-CO-Nterm1049 (SEQ ID NO:59), hSCN1A-CO-Nterm956 (SEQ ID NO:61), or hSCN1A-CO-Nterm947 (SEQ ID NO:63). In certain embodiments, the C-terminal portion of the coding sequence for SCN1A comprises hSCN1A-CO-Cterm949 (SEQ ID NO:60), hSCN1A-CO-Cterm1042 (SEQ ID NO:62), or hSCN1A-CO-Cterm1051 (SEQ ID NO:64).

[0068] Exemplary reporter genes / proteins include those expressed by Addgene ID No. 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), ID No. 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), ID No. 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), ID No. 83898 (pAAV-mDlx-ChR2-mCherry-Fishell-3), ID No. 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), ID No. 83900 (pAAV-mDlx-GFP-Fishell-1), and ID No. 89897 (pcDNA3-FLAG-mTET2(N500)). Exemplary reporter genes include, among others, expressible fluorescent proteins or expressible biotin; blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen), CopGFP, AceGFP, avGFP, ZsGreen1, Oregon Green, etc.) TM (Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato, dTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red) TM(Thermo Fisher Scientific)); far-red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellow1); or reporter genes encoding tandemly linked complexes.

[0069] GFP, a 238-amino acid (26.9 kDa) protein originally isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea, fluoresces green when exposed to blue light. The GFP isolated from A. victoria has a major excitation peak at 395 nm and a minor excitation peak at 475 nm. Its emission peak is at 509 nm, in the low-green region of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. Due to its wide range of applications and the demand for further improvements, various GFP variants have been created. The first major improvement was a single point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the spectral properties of GFP, increasing its fluorescence and photostability, and shifting its major excitation peak to 488 nm while maintaining its emission peak at 509 nm. Enhanced GFP (EGFP) was obtained by adding a point mutation (F64L) to GFP that improves folding efficiency at 37 °C. EGFP has an extinction coefficient (ε) of 55,000 L / mol cm, which corresponds to a 9.13 × 10 per molecule extinction coefficient. -21 m 2 Also known as the optical cross section of a protein, Superfolder GFP was reported in 2006 as a series of GFP variants that can rapidly fold and mature even when fused to poorly folded peptides.

[0070] "Yellow fluorescent protein" (YFP) is a genetic variant of green fluorescent protein derived from Aequorea victoria. Its excitation peak is at 514 nm and its emission peak is at 527 nm.

[0071] In certain embodiments, the artificial expression constructs of the present disclosure may include DNA editing tools such as CRISPR / Cas (e.g., guide RNAs and nucleases such as Cas, Cas9, cpf1, etc.) and RNA editing tools. Additionally, the functional molecule may include recombinant Cpf1 as described in U.S. Patent Publication No. 2018 / 0030425, U.S. Patent Publication No. 2016 / 0208243, WO / 2017 / 184768, and Zetsche et al. (2015) Cell 163: 759-771; single-stranded gRNA (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563), an editase, a guide RNA molecule, a microRNA, or a homologous recombination donor cassette.

[0072] In certain embodiments, the artificial expression constructs of the present disclosure may include a localization cassette. In certain embodiments, the localization cassette is used to localize molecules (e.g., vectors, proteins, sensors) to specific subcellular compartments, such as the cell body, axons, or dendrites of a neuron. In certain embodiments, the localization cassette includes a somatic tag (e.g., somatic (EE-RR)) for somatic localization; an axonal tag (e.g., derived from GAP43) or synaptophysin (sy) for axonal localization; a hydrophobic tail for plasma membrane localization; and a hydrophobic or alkyl chain for endoplasmic reticulum localization. In certain embodiments, the localization cassette is fused to a sensor molecule, such as a GECI. In certain embodiments, fusion proteins of the localization cassette and a GECI include somatic-jGCaMP8s, axon-jRGECO1a, syGCaMP5G, and somatic-jGCaMP7s. The artificial expression constructs of the present disclosure may encode nuclear transport proteins such as histone H1, histone H2A, histone H2B, histone H3, histone H4, histone-like proteins HPhA and H2B*.

[0073] In certain embodiments, the artificial expression constructs of the present disclosure may comprise a tag cassette. Examples of tag cassettes include His tag (HHHHHH; SEQ ID NO: 34), Flag tag (DYKDDDDK; SEQ ID NO: 35), Xpress tag (DLYDDDDK; SEQ ID NO: 36), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 37), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 38), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 39), Myc tag (EQKLISEEDL; SEQ ID NO: 40), Strep tag (meaning the original STREP® tag) (WRHPQFGG; SEQ ID NO: 41), STREP tag II (WSHPQFEK; SEQ ID NO: 42; (Institut fur Bioanalytik (IBA) GmbH, Germany; see, e.g., U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 43), Softag In certain embodiments, the tag cassette includes a fusion tag cassette such as 3XFLAG. In certain embodiments, the 3XFLAG includes the sequence shown in SEQ ID NO: 15.

[0074] Additional effector elements include Cre, iCre, dgCre, FlpO, and tTA2. iCre refers to codon-improved Cre. dgCre is a GFP / Cre recombinase fusion gene enhanced by the N-terminal fusion of the first 159 amino acids of the dihydrofolate reductase gene (DHFR or folA) from the E. coli K12 chromosome, carrying the G67S mutation and the destabilization domain mutation R12Y / Y100I via recombination. FlpO is a codon-optimized form of FLPe, which significantly improves protein expression and FRT recombination efficiency in mouse cells. The FLP / FRT system, like the Cre / LoxP system, is widely used for gene expression (and the generation of conditional knockout mice using the FLP / FRT system is also widely used). tTA2 refers to the tetracycline transactivator.

[0075] An exemplary self-cleaving peptide is the 2A peptide, which allows two proteins to be produced from a single mRNA. 2A sequences are short (e.g., 20 amino acids long) and are often used in size-constrained constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the artificial expression construct of the present disclosure includes an internal ribosome entry site (IRES) sequence. The IRES can initiate ribosome translation from a second internal site on the mRNA molecule, allowing two proteins to be produced from a single mRNA. In certain embodiments, the IRES includes IRES2. In certain embodiments, IRES2 enables the translation of a second protein open reading frame (ORF) from a single transcript. This feature of IRES2 differs from 2A sequences, which can cleave a single ORF into two proteins with similar production efficiency as IRES2.

[0076] Coding sequences encoding the molecules (e.g., RNAs and proteins) described herein can be obtained from publicly available databases or publications. Coding sequences may further contain various sequence polymorphisms, mutations, and / or sequence variants, where such changes do not affect the function of the encoded molecule. The term "encoding" refers to the property of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, or mRNA, to serve as a template for the synthesis of other molecules, such as proteins.

[0077] The term "gene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or post-transcriptional regulatory elements (e.g., termination regions). Furthermore, the term may include any introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. These sequences may further include degenerate codons of a reference sequence or sequences that may be introduced to confer codon preference in a particular type of organism or cell.

[0078] Promoters include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also include strong promoters, weak promoters, constitutive promoters, and / or inducible promoters. Inducible promoters induce expression in response to specific conditions, signals, or cellular events. For example, a promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to induce transcription from the promoter. Specific examples of promoters include minBglobin (also called minBGprom), CMV promoter, hSyn1 promoter, truncated hSyn1 promoter, minCMV, minCMV* (minCMV* is minCMV with the SacI restriction site removed), minRho, minRho* (minRho* is minRho with the SacI restriction site removed), SV40 immediate early promoter, Hsp68 minimal promoter (proHSP68), and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. A minimal promoter does not have the activity to induce gene expression by itself, but when linked to a nearby enhancer element, it can be activated and induce gene expression.

[0079] In certain embodiments, the expression construct is provided in a vector. The term "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is typically linked to, e.g., inserted into, the vector's nucleic acid molecule. The vector may contain a sequence that induces autonomous replication in the cell or may contain a sequence that allows integration into the host cell's DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids and RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0080] The term "viral vector" is used broadly to refer to a nucleic acid molecule that contains virus-derived components that facilitate the transfer and expression of non-naturally occurring nucleic acid molecules in cells. The term "adeno-associated viral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from AAV. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from lentiviruses, etc. The term "hybrid vector" refers to a vector that contains structural elements and / or functional genetic elements derived from two or more viruses.

[0081] "Adenoviral vector" refers to a construct containing sufficient adenoviral sequences (a) to facilitate packaging of an artificial expression construct and (b) to express a cloned coding sequence in either sense or antisense orientation. Recombinant adenoviral vectors include genetically engineered forms of adenovirus. Adenovirus is a 36 kb linear, double-stranded DNA virus, allowing up to 7 kb of foreign sequence to replace a large portion of the adenoviral DNA. Adenoviral DNA replicates episomally without causing genotoxicity; unlike retroviruses, adenoviral infection of host cells does not result in chromosomal integration. Furthermore, adenoviruses are structurally stable, and no genome rearrangements have been observed after extensive amplification.

[0082] Adenoviruses are particularly suitable for use as gene transfer vectors due to their moderate genome size, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the adenovirus genome contain 100-200 base pair inverted repeats (ITRs), which are cis-receptors required for viral DNA replication and packaging. The early (E) and late (L) regions of the adenovirus genome contain various transcription units that are divided by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the adenoviral genome and several cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and shut-off of host cell protein biosynthesis. Late gene products, including most of the adenovirus capsid proteins, are expressed only after significant processing of a single primary transcript driven by the major late promoter (MLP). MLP is particularly efficient during the late stages of infection, and all mRNAs driven by this promoter have a tripartite 5'-leader (TPL) sequence, which is preferentially selected by the mRNA for translation.

[0083] Other than the requirement that the adenoviral vector be replication-deficient or at least conditionally-deficient, the characteristics of the adenoviral vector are not believed to be critical to successfully practicing certain embodiments disclosed herein. The adenovirus may be of any of the 42 known serotypes or subgenera A-F. In certain embodiments, adenovirus of serotype 5 of the C subgenus is preferred as the starting material for obtaining conditional replication-deficient adenoviral vectors for use in certain embodiments, since adenovirus type 5 is the human adenovirus for which the most biochemical and genetic information is known and has historically been used in the majority of adenovirus vector constructions.

[0084] As described herein, typical vectors are replication-deficient and lack the adenovirus E1 region. Therefore, it is most convenient to introduce a polynucleotide encoding a gene of interest into the site from which the coding sequence of the E1 region has been deleted. However, the insertion position of the construct within the adenovirus sequence is not critical. The polynucleotide encoding a gene of interest can be inserted into the deleted E3 region of an E3 replacement vector or into the E4 region, with the E4 region deficiency complemented by a helper cell line or helper virus.

[0085] Adeno-associated virus (AAV) is a parvovirus found as a contaminant in adenovirus stocks. AAV is a ubiquitous virus (85% of the US population possesses anti-AAV antibodies) and does not cause disease. Furthermore, AAV replication is dependent on the presence of a helper virus (e.g., adenovirus), and therefore AAV is classified as a dependovirus. Various serotypes have been isolated, of which AAV-2 is the most extensively characterized. AAV contains single-stranded linear DNA, which is packaged by the capsid proteins VP1, VP2, and VP3 to form icosahedral virions with a diameter of 20–24 nm.

[0086] The AAV DNA is 4.7 kilobases long. It contains two open reading frames flanked by two ITRs. The AAV genome contains two main genes: rep and cap. The rep gene encodes the proteins responsible for AAV viral replication, while the cap gene encodes the capsid proteins VP1-VP3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only cis-terminal repeat components of AAV required for chromosomal integration. Therefore, AAV can be used as a vector, allowing the complete viral coding sequence to be removed and replaced with a gene cassette for delivery. Three AAV viral promoters have been identified, named p5, p19, and p40, based on their map locations. Transcription from p5 and p19 leads to the production of the rep protein, while transcription from p40 leads to the production of the capsid protein.

[0087] AAV is outstanding for use in the present disclosure because it has an excellent safety profile and can be expressed in target cell populations by modifying the capsid and genome. scAAV refers to self-complementary AAV. pAAV refers to plasmid adeno-associated virus. rAAV refers to recombinant adeno-associated virus. pSMART-HCKan is a high copy number vector with a kanamycin resistance marker for efficient blunt-end cloning of unstable sequences.

[0088] Other viral vectors may also be used, such as those derived from viruses such as vaccinia virus, poliovirus, and herpesvirus, which offer beneficial characteristics for a variety of mammalian cells.

[0089] Retroviruses are commonly used as tools for gene delivery. Retroviruses are RNA viruses whose genomic RNA is reverse transcribed to produce a double-stranded linear DNA copy, which is then covalently integrated into the host genome. Once integrated into the host genome, the retrovirus is called a provirus. The provirus serves as a template for RNA polymerase II, inducing the expression of RNA molecules encoding the structural proteins and enzymes required for the production of new virus particles.

[0090] Examples of retroviruses suitable for use in certain embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0091] "Lentivirus" refers to the complex retrovirus group (or genus). Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV types 1 and 2); visna-maedi virus (VMV); caprine arthritis-encephalomyelitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In certain embodiments, an HIV-based vector backbone (i.e., cis-acting sequence elements of HIV) can be used.

[0092] In some types of vectors, safety can be improved by replacing the U3 region of the 5'LTR, which drives transcription of the viral genome during viral particle production, with a heterologous promoter. Examples of heterologous promoters that can be used for this purpose include the simian virus 40 (SV40) (e.g., early or late) promoter, the cytomegalovirus (CMV) (e.g., immediate early) promoter, the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, and the herpes simplex virus (HSV) (thymidine kinase) promoter. Conventional promoters can drive high-level transcription independent of Tat. Replacement of the U3 region with a heterologous promoter eliminates the entire U3 sequence from the viral production system, thereby reducing the likelihood of recombination leading to the production of replicative virus. In certain embodiments, a heterologous promoter offers the additional advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter may be an inducible promoter, such that all or part of the viral genome is transcribed only in the presence of an inducer. Inducers include one or more compounds or physiological conditions, such as the temperature or pH of the culture of the host cells.

[0093] In certain embodiments, the viral vector comprises a TAR element. "TAR" refers to the "transactivation response" gene element present in the R region of the LTR of lentivirus. This element interacts with the transactivator (tat) gene element of lentivirus to enhance viral replication. However, this element is not required in embodiments in which the U3 region of the 5'LTR is replaced with a heterologous promoter.

[0094] The "R region" refers to the region within the retroviral LTR from the start of the cap site (i.e., the transcription initiation site) to just before the start of the poly(A) tail. The R region is also defined as the region between the U3 and U5 regions. The R region plays a role in moving nascent DNA from one end of the genome to the other during reverse transcription.

[0095] In certain embodiments, the expression of heterologous sequences in viral vectors can be increased by incorporating posttranscriptional regulatory elements and efficient polyadenylation sites into the viral vector, and a transcription termination signal may also be incorporated into the viral vector. Various posttranscriptional regulatory elements can increase the expression of heterologous nucleic acids. Examples of posttranscriptional regulatory elements include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the hepatitis B virus posttranscriptional regulatory element (HPRE) (Smith et al., Nucleic Acids Res. 26(21):4818-4827, 1998); and other posttranscriptional regulatory elements (Liu et al., Genes Dev., 9:1766, 1995). In certain embodiments, vectors contain posttranscriptional regulatory elements such as WPRE and HPRE. In certain embodiments, vectors lack or do not contain posttranscriptional regulatory elements such as WPRE and HPRE.

[0096] Expression of heterologous genes can be increased by elements that can induce efficient transcription termination and polyadenylation of heterologous nucleic acid transcripts. Transcription termination signals are typically found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation signal at the 3' end of a polynucleotide encoding an expressed molecule (e.g., a protein). A "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that induces both transcription termination and polyadenylation of a nascent RNA transcript transcribed by RNA polymerase II. A polyadenylation sequence can improve mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thereby contributing to improved translation efficiency. In certain embodiments, BGHpA, hGHpA, SV40pA, or shortPolyA may be utilized. In certain embodiments, a preferred embodiment of an expression construct includes a terminator element. The terminator element can increase the amount of transcription and minimize read-through transcription from the construct to other plasmid sequences.

[0097] In certain embodiments, the viral vector further comprises one or more insulator elements. The insulator elements may protect sequences expressed from the viral vector, such as effector elements or expressible elements, from integration site effects. Integration site effects occur through cis-acting elements within genomic DNA, meaning that the imported sequence may or may not be expressed (i.e., position effects; see, for example, Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum. Genet., 109:471, 2001). In certain embodiments, the viral import vector comprises one or more insulator elements in the 3'LTR, and upon provirus integration into the host genome, the insulator is integrated into both the 5'LTR and the 3'LTR during replication of the 3'LTR. Insulators suitable for use in certain embodiments include the chicken β-globin insulator (see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), the SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer-blocking insulators (Liu et al., Nature Biotechnology, 33:198, 2015).

[0098] In addition to those described above, various types of suitable expression vectors are known to those skilled in the art. These known expression vectors include commercially available expression vectors designed for general recombinant manipulation, such as plasmids containing one or more reporter genes and the regulatory elements required for cellular expression of the reporter genes. Many vectors are commercially available from companies such as Invitrogen, Stratagene, and Clontech, and are described in various accompanying guidebooks. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct capable of expressing an encoded gene in mammalian cells, such as pUC plasmids and Bluescript plasmids.

[0099] Particular embodiments of the vectors disclosed herein include those set forth in the table below. [Table 1]

[0100] Subcomponent sequences within a larger vector sequence can be readily identified by one of skill in the art based on the present disclosure. Nucleotides between the identifiable subcomponents listed in the table above are restriction enzyme recognition sites used in construct assembly (cloning) and, in some cases, additional nucleotides with no identifiable function. These segments of the complete vector sequence can be adjusted using various cloning techniques and / or various vectors. Short palindromic sequences of six bases typically represent vector construction artifacts that are not critical to vector function.

[0101] In certain embodiments, a vector (e.g., AAV) is selected that has a capsid that can cross the blood-brain barrier (BBB). In certain embodiments, the vector is modified to contain a capsid that crosses the blood-brain barrier. Examples of AAVs with viral capsids that can cross the blood-brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014; 7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014; 22(7): 1299-1309), AAV1R6, AAV1R7 (Albright et al., Mol Ther. 2018; 26(2): 510), rAAVrh.8 (Yang et al., supra), AAV-BR1 (Marchio et al., EMBO Mol Med. 2016; 8(6): 592), AAV-PHP.S (Chan et al., Nat Neurosci. 2017; 20(8): 1172), and AAV-PHP.B (Deverman et al., Nat Biotechnol. 2016; 34(2): 204), AAV-PPS (Chen et al., Nat Med. 2009; 15: 1215), and PHP.eB. In certain embodiments, the capsid of PHP.eB differs from that of AAV9 in that, when comparing AAV9 as a reference, amino acid residues 586 and beyond, S-AQ-A (SEQ ID NO: 46), are changed to S-DGTLAVPFK-A (SEQ ID NO: 47). In certain embodiments, PHP.eb refers to the sequence of SEQ ID NO: 30.

[0102] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the blood-brain barrier (BBB) ​​upon intravenous injection. AAV9 transduces a wide range of areas in the central nervous system (CNS), allowing for minimally invasive treatment (Naso et al., BioDrugs. 2017; 31(4): 317). Examples of this have been reported in clinical trials, such as AveXis' AVXS-101 (NCT03505099) for the treatment of spinal muscular atrophy (SMA) syndrome and in clinical trials for the treatment of CLN3-associated neuronal ceroid lipofuscinosis (NCT03770572).

[0103] AAVrh.10, an AAV originally isolated from rhesus macaques, has weak human seroreactivity compared to other common serotypes used in gene delivery applications (Selot et al., Front Pharmacol. 2017;8:441) and has been evaluated in several clinical trials (LYS-SAF302, LYSOGENE, and NCT03612869).

[0104] AAV1R6 and AAV1R7 are two variants isolated from a library of chimeric AAV vectors (in which the capsid domain of AAVrh.10 is replaced with that of AAV1) that retain the ability to cross the BBB and transduce the central nervous system, but exhibit significantly reduced transduction of the liver and vascular endothelium.

[0105] rAAVrh.8 is also an AAV isolated from rhesus macaques that, upon peripheral administration, exhibits widespread transduction of glial and neuronal cells in clinically relevant areas, with reduced peripheral tissue tropism compared to other vectors.

[0106] AAV-BR1 is an AAV2 variant that displays the NRGTEWD ​​epitope (SEQ ID NO: 48) and was isolated during in vivo screening of a random AAV-display peptide library. AAV-BR1 exhibits high specificity with high transgene expression in the brain and minimizes off-target affinity (including affinity for the liver) (Korbelin et al., EMBO Mol Med. 2016; 8(6): 609).

[0107] AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence QAVRTSL (SEQ ID NO: 49) and transduces neurons of the enteric nervous system and potently transduces peripheral sensory afferents projecting to the spinal cord and brainstem.

[0108] AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated by the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO: 50). AAV-PHP.B transfers genes throughout the central nervous system more efficiently than AAV9, transducing a large proportion of astrocytes and neurons in multiple central nervous system regions.

[0109] AAV-PPS is an AAV2 variant created by inserting the DSPAHPS epitope (SEQ ID NO: 51) into the capsid of AAV2, and exhibits dramatically improved brain tropism compared to AAV2.

[0110] For more information regarding capsids crossing the blood-brain barrier, see Chan et al., Nat. Neurosci. 2017 Aug: 20(8): 1172-1179.

[0111] In certain embodiments, capsids are selected that transduce target cell types in primates after administration (e.g., intravenous administration). In certain embodiments, capsids are selected that transduce tissues and cell types affected by SCN1A deletions broadly after administration to these tissues and cell types. In certain embodiments, the target cell types are neurons. In certain embodiments, neurons include GABAergic neurons or glutamatergic neurons. In certain embodiments, GABAergic neurons include whole GABAergic neurons, GABAergic neurons in the forebrain, GABAergic neurons in the hippocampus, or GABAergic neurons in the cortex. In certain embodiments, glutamatergic neurons include glutamatergic neurons in the forebrain.

[0112] (ii) Composition for Administration The disclosed artificial expression constructs and vectors (herein referred to as bioactive components) that rescue voltage-gated sodium channels can be formulated in a single carrier or in two or more carriers suitable for administration to cells, tissue slices, animals (e.g., mice or non-human primates), or humans. The bioactive components included in the compositions described herein can be formulated as neutral forms, free bases, or pharmacologically acceptable salts.

[0113] Pharmaceutically acceptable salts include the acid addition salts (formed from the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, etc. Also, salts formed with the free carboxyl groups can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, or such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0114] Carriers for physiologically active ingredients include solvents, dispersion media, vehicles, coating agents, diluents, isotonicity agents, absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is well known in the art. Except insofar as a conventional media or agent is incompatible with the physiologically active ingredients of the present invention, any conventional media or agent can be used in combination with the compositions described herein.

[0115] The term "pharmaceutically acceptable carrier" refers to a carrier that does not produce an allergic or similar untoward reaction when administered to a human, in particular in an embodiment, when administered intravenously.

[0116] In certain embodiments, the compositions of the present invention can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular (ICV), intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or for direct injection or administration into one or more cells, tissues, or organs.

[0117] The compositions of the present invention may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0118] The formation and use of liposomes are widely known to those skilled in the art. Liposomes have been developed to improve serum stability and blood half-life (see, for example, U.S. Patent No. 5,741,516). Furthermore, various methods have been reported for using liposomes and liposome-like preparations as potential drug carriers (see, for example, U.S. Patent No. 5,567,434; U.S. Patent No. 5,552,157; ​​U.S. Patent No. 5,565,213; U.S. Patent No. 5,738,868; and U.S. Patent No. 5,795,587).

[0119] The present disclosure also provides pharmaceutically acceptable nanocapsule formulations of the bioactive ingredients of the present invention. Generally, nanocapsule formulations can encapsulate compounds in a stable and reproducible manner (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res. 15(7):1056-1062, 1998; Quintanar-Guerrero et al., J. Microencapsul. 15(1):107-119, 1998; Douglas et al., Crit Rev Ther Drug Carrier Syst 3(3):233-261, 1987). To avoid side effects caused by large amounts of macromolecules being taken up into cells, such nanoparticles can be designed using polymers that can be degraded in vivo. The present disclosure also envisages the use of biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements.This type of microparticle can be easily produced, and is described, for example, in Couvreur et al., J Pharm Sci 69(2):199-202,1980; Couvreur et al., Crit Rev Ther Drug Carrier Syst. 5(1)1-20,1988; zur Muhlen et al., Eur J Pharm Biopharm, 45(2):149-155,1998; Zambaux et al., J Control Release 50(1-3):31-40,1998; and United States Patent No. 5,145,684.

[0120] Injectable compositions include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468). Injectable compositions delivered by injection are in a sterile fluid form sufficient to be delivered using a syringe. In certain embodiments, injectable compositions are typically stable during manufacturing and storage and may contain one or more preservative compounds to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, which may contain, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof, and / or vegetable oils. To maintain proper fluidity, a coating such as lecithin may be used, or in the case of dispersions, a particle size may be maintained to the required size, and / or a surfactant may be used. To prevent the action of microorganisms, various antibacterial and / or antifungal agents may be used, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the injectable composition contains an isotonic agent, such as sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by incorporating an agent that delays absorption, such as aluminum monostearate or gelatin, into the injectable composition. If necessary, an appropriate buffer may be added to the injectable composition, and the diluted liquid is first made isotonic with sufficient saline or glucose.

[0121] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, or mixtures thereof, or oils. As noted herein, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0122] Sterile compositions can be prepared by mixing the physiologically active ingredient with any other ingredients (e.g., those listed above) in the appropriate amount of solvent and then sterile filtering. Dispersions are typically prepared by dispersing the various sterilized physiologically active ingredients in a sterile solvent containing a basic dispersion medium and the other required ingredients (e.g., those listed above). In the case of sterile powders for preparing sterile injectable solutions, a preferred method is to first sterile filter a solution containing the physiologically active ingredient and the other desired ingredients, and then vacuum-dry or freeze-dry the solution to prepare a powder containing the physiologically active ingredient and the other desired ingredients.

[0123] Oral compositions may be in liquid form, such as solutions, syrups, or suspensions, and may be provided as pharmaceutical products to be reconstituted with water or other suitable solvents before use. Such liquid preparations may be prepared by conventional methods using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, ester oil, or fractionated vegetable oil); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, or sorbic acid). The compositions of the present invention may be prepared in the form of tablets or capsules by conventional methods using pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods known in the art.

[0124] Compositions for inhalation can be delivered in the form of an aerosol spray from a pressurized pack or a nebulizer, using a suitable propellant, such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The compositions may also be formulated into capsules or cartridges (e.g., gelatin capsules or cartridges) for use in an inhaler or nebulizer, which contain a powder mix of the compositions described herein and a suitable powder base, such as lactose or starch.

[0125] Additionally, compositions of the present invention include microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Pat. No. 5,697,899).

[0126] Supplementary active ingredients can also be incorporated into the compositions of the present invention.

[0127] Typically, the compositions of the present invention contain at least 0.1% or more of the physiologically active ingredient, although the percentage of the physiologically active ingredient may vary, conveniently ranging from 1% or 2% to 70% or 80% or more, or even from 0.5 to 99%, based on the total weight or volume of the compositions of the present invention. Naturally, the amount of physiologically beneficial physiologically active ingredient in each composition may be adjusted to provide an appropriate dosage for a given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art in preparing such pharmaceutical formulations, and various compositions and dosages may be desirable accordingly.

[0128] In certain embodiments, for human administration, compositions of the invention must meet sterility, pyrogenicity, general safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other responsible regulatory authorities in other countries.

[0129] (iii) Cell lines containing the artificial expression constructs The present disclosure includes cells comprising the artificial expression constructs described herein. Cells transformed with the artificial expression constructs can be used for a variety of purposes, such as neuroanatomical studies, evaluation of functional and / or non-functional proteins, and drug screening to evaluate the regulatory properties of enhancers.

[0130] While a variety of host cell lines can be used, in certain embodiments, the host cell is a mammalian cell. In certain embodiments, the artificial expression constructs of the present disclosure contain DLX2.0, minBglobin promoter, hSyn1 promoter, CMV promoter, truncated hSyn1 promoter, 4x2C miR binding site, 8x2C and the cell line comprises a miR binding site, eHGT_078h, Cfa-N, Cfa-C, hSCN1A-CO-Nterm1049, hSCN1A-CO-Cterm949, hSCN1A-CO-Nterm956, hSCN1A-CO-Cterm1042, hSCN1A-CO-Nterm947, and / or hSCN1A-CO-Cterm1051, and / or a regulatory element and / or vector sequence selected from CN3252, CN3254, CN3683, CN3684, CN3251, CN3253, CN3677, CN3678, CN4541, CN4542, CN4217, CN4218, CN4642, and CN4643, and the cell line is a human cell, a primate cell, or a mouse cell. Additionally, cell lines that can be utilized for gene transfer in the present disclosure include primary cell lines derived from living tissues such as rat or mouse brain, and organotypic cell cultures such as brain slices derived from animals such as rat, mouse, non-human primate, or human neurosurgical tissue.

[0131] WO91 / 13150 describes various cell lines, including neuronal cell lines, and methods for their production. Similarly, WO97 / 39117 describes neuronal cell lines and methods for producing such cell lines. The neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.

[0132] In certain embodiments, the term "neuron" is used to describe any neuronal cell, anything related to or including a neuronal cell. Neuronal cells are defined by the characteristic of having an axon and dendrites. The term "neuron-specific" refers to something that is found in neuronal cells or cells derived therefrom but is not found or is substantially absent in cells that are not derived from neuronal cells or in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes); or activity that occurs in neuronal cells or cells derived therefrom but is not found or is substantially absent in cells that are not derived from neuronal cells or in non-neuronal cells (e.g., glial cells such as astrocytes and oligodendrocytes).

[0133] In certain embodiments, non-neuronal cell lines such as mouse embryonic stem cells may be used. Cultured mouse embryonic stem cells can be transiently transfected with a plasmid construct to analyze the expression of the gene construct. Mouse embryonic stem cells are pluripotent, undifferentiated cells. Mouse embryonic stem cells can be maintained in an undifferentiated state by leukemia inhibitory factor (LIF). Differentiation of mouse embryonic stem cells can be induced by removing LIF. Mouse embryonic stem cells form various types of differentiated cells in culture. Differentiation of mouse embryonic stem cells occurs through the expression of tissue-specific transcription factors, which allows for evaluation of the function of enhancer sequences (see, for example, Fiskerstrand et al., FEBS Lett 458: 171-174, 1999).

[0134] A method for differentiating stem cells into neuronal cells involves replacing the stem cell culture medium with a medium containing basic fibroblast growth factor (bFGF), heparin, N2 supplements (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin, and polyornithine. A method for producing myelin-forming oligodendrocytes from stem cells is described in Hu et al., 2009, Nat. Protoc. 4:1614-22. Bibel et al., 2007, Nat. Protoc. 2:1034-43, describes a protocol for producing glutamatergic neurons from stem cells, and Chatzi et al., 2009, Exp. Neurol. 217:407-16, describes a procedure for producing GABAergic neurons. This procedure involves exposing stem cells to all-trans retinoic acid for 3 days. Next, by culturing in a serum-free neuronal induction medium such as neurobasal medium supplemented with B27, bFGF, and EGF, GABAergic neurons, which account for 95% of all cells, are obtained.

[0135] U.S. Patent Publication No. 2012 / 0329714 describes the use of prolactin to increase neural stem cell numbers, and U.S. Patent Publication No. 2012 / 0308530 describes a culture surface with amino groups that promotes neuronal differentiation into neurons, astrocytes, and oligodendrocytes. Thus, the fate of neural stem cells can be controlled by various extracellular factors. Commonly used extracellular factors include brain-derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425); fibroblast growth factor (bFGF; U.S. Patent No. 5,766,948; FGF-1, FGF-2); neurotrophin 3 (nt-3) and neurotrophin 4 (nt-4) (Caldwell, et al., 2001, Nat. Biotechnol. 1;19:475-9); ciliary neurotrophic factor (CNTF); BMP-2 (U.S. Patent Nos. 5,948,428 and 6,001,654); isobutyl-3-methylxanthine; leukemia growth inhibitory factor (LIF; U.S. Patent No. 6,103,530); somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate); epidermal growth factor (EGF); dexamethasone (a glucocorticoid hormone); forskolin; ligands for GDNF family receptors; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transforming growth factor alpha and TGF-beta (U.S. Patent Nos. 5,851,832 and 5,753,506).

[0136] In certain embodiments, the yeast one-hybrid system may be used to identify compounds that inhibit specific protein-DNA interactions, such as those of the DLX2.0, minBglobin promoter, hSyn1 promoter, CMV promoter, truncated hSyn1 promoter, 4x2C miR binding site, 8x2C miR binding site, and / or eHGT_078h transcription factor.

[0137] Transgenic animals are described below. Cell lines may be derived from such transgenic animals. For example, cell lines carrying artificial expression constructs integrated into their genomes can be obtained from primary tissue cultures derived from transgenic mice (e.g., as described below) (see, e.g., MacKenzie & Quinn, Proc Natl Acad Sci USA 96: 15251-15255, 1999).

[0138] (iv) Transgenic animals Another aspect of the present disclosure includes transgenic animals comprising in their genome an artificial expression construct comprising DLX2.0, a minBglobin promoter, an hSyn1 promoter, a CMV promoter, a truncated hSyn1 promoter, a 4x2C miR binding site, an 8x2C miR binding site, eHGT_078h, Cfa-N, Cfa-C, hSCN1A-CO-Nterm1049, hSCN1A-CO-Cterm949, hSCN1A-CO-Nterm956, hSCN1A-CO-Cterm1042, hSCN1A-CO-Nterm947, and / or hSCN1A-CO-Cterm1051 operably linked to a heterologous coding sequence. In certain embodiments, the genome of the transgenic animal comprises a combination of CN3252 and CN3254, a combination of CN3683 and CN3684, a combination of CN3251 and CN3253, a combination of CN3677 and CN3678, a combination of CN4541 and CN4542, a combination of CN4217 and CN4218, or a combination of CN4642 and CN4643. In certain embodiments, when a non-integrating vector is utilized, the transgenic animal comprises a combination of DLX2.0, a minB globin promoter, a hSyn1 promoter, a CMV promoter, a truncated hSyn1 promoter, a 4x2C miR binding site, an 8 ... The one or more cells comprise an artificial expression construct comprising a miR binding site, eHGT_078h, Cfa-N, Cfa-C, hSCN1A-CO-Nterm1049, hSCN1A-CO-Cterm949, hSCN1A-CO-Nterm956, hSCN1A-CO-Cterm1042, hSCN1A-CO-Nterm947, and / or hSCN1A-CO-Cterm1051, and / or a combination of CN3252 and CN3254, a combination of CN3683 and CN3684, a combination of CN3251 and CN3253, a combination of CN3677 and CN3678, a combination of CN4541 and CN4542, a combination of CN4217 and CN4218, or a combination of CN4642 and CN4643.

[0139] A detailed description of methods for producing transgenic animals is provided in U.S. Patent No. 4,736, 866. The transgenic animal may be of any non-human species, preferably a non-human primate (NHP), sheep, horse, cow, pig, goat, dog, cat, rabbit, chicken, or rodent, such as a guinea pig, hamster, gerbil, rat, mouse, or ferret.

[0140] In certain embodiments, by producing transgenic animals, organisms can be obtained in which recombinant constructs are introduced into the same genome integration site in every cell.Therefore, the cell lines derived from such transgenic animals have consistent characteristics in that they have recombinant constructs in the same genome integration site in every cell, and therefore, all of these cells undergo the same variegated position effect.In contrast, when genes are introduced into cell lines or primary cell cultures, heterologous expression of constructs can be obtained.This method has the disadvantage that the expression of introduced DNA can be affected by the specific genetic background of host animals.

[0141] As discussed above in connection with cell lines, the artificial expression constructs of the present disclosure can be used to genetically modify mouse embryonic stem cells using techniques known in the art. Typically, the artificial expression constructs are introduced into cultured mouse embryonic stem (ES) cells. The transformed ES cells are then injected into blastocysts derived from a host mother, and the host embryo is reimplanted into the host mother. This procedure results in chimeric mice with tissues composed of cells derived from both the embryonic stem cells present in the cultured cell line and the embryonic stem cells present in the host embryo. Typically, mice from which cultured ES cells are isolated for gene transfer are selected to have a coat color different from that of the host mouse into whose embryo the transformed cells are injected. Thus, the chimeric mice have a mixed coat color. If at least a portion of the germline tissue is derived from the genetically modified cells, the chimeric mice can then be bred with an appropriate strain to obtain offspring carrying the transgene.

[0142] In addition to the delivery methods described above, other methods of delivering artificial expression constructs to target cells or tissues or organs of animals, particularly cells, organs, or tissues of mammalian vertebrates, are contemplated, including sonophoresis (e.g., ultrasound, as described in U.S. Pat. No. 5,656,016); intraosseous injection (U.S. Pat. No. 5,779,708); microchip devices (U.S. Pat. No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1):33-58, 1998); transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208); feedback-controlled delivery (U.S. Pat. No. 5,697,899), as well as other delivery methods available and / or described elsewhere in this disclosure.

[0143] (v) How to use In certain embodiments, administering a composition comprising the bioactive component described herein to a subject results in targeted expression of a protein or nucleotide sequence that rescues the function of a voltage-gated sodium channel in a target cell type in the subject.The subject may be an isolated cell, a cell network, a tissue section, an experimental animal, a veterinary animal, or a human.In certain embodiments, rescuing the function of a voltage-gated sodium channel includes converting a subject's cells that lack sufficient amounts of Nav1.1 sodium channels and / or lack sufficient activity of Nav1.1 sodium channels into cells that express sufficient amounts and sufficient activity of exogenous voltage-gated sodium channels, thereby restoring neuronal function and preventing epileptiform circuit activity.

[0144] In certain embodiments, rescue of voltage-gated sodium channel function is evidenced by one or more of an increase in sodium channel current in target neurons genetically modified with the bioactive component and / or an increase in excitability of target neurons genetically modified with the bioactive component. In certain embodiments, rescue of voltage-gated sodium channel function is evidenced by one or more of an increase in sodium channel conductance in cells of a target type genetically modified with the bioactive component and / or an increase in sodium channel influx in response to voltage depolarization of cells of a target type genetically modified with the bioactive component. The increase may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Inhibitory neuron output can be measured using electrophysiological methods such as multi-electrode arrays and patch clamping.

[0145] In certain embodiments, the artificial expression constructs of the present disclosure are expressed in any neuron. In certain embodiments, the artificial expression constructs of the present disclosure are expressed in a cell line. In certain embodiments, the artificial expression constructs of the present disclosure are expressed in a target cell type. In certain embodiments, the target cell type is a neuron. In certain embodiments, the neuron comprises a GABAergic neuron or a glutamatergic neuron. In certain embodiments, the GABAergic neuron is whole GABAergic neuron, forebrain GABAergic neuron, hippocampal GABAergic neuron, or cortical GABAergic neuron. In certain embodiments, the glutamatergic neuron comprises forebrain glutamatergic neuron.

[0146] In certain embodiments, rescue of voltage-gated sodium channel function is evidenced by an increase in sodium current-dependent fast firing in forebrain interneurons, e.g., using a mouse model. In certain embodiments, rescue of voltage-gated sodium channel function is evidenced by a delay or prevention of heat-induced seizures in a mouse model, as described herein.

[0147] Certain embodiments include identifying a subject with an SCN1A-associated seizure disorder in a target cell type (e.g., GABAergic neurons of the forebrain). Such subjects can be identified based on a diagnosis of a disorder associated with an SCN1A-associated seizure disorder, including Dravet syndrome, myoclonic seizures, myoclonic-astatic epilepsy (MAE), refractory childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epilepsy with febrile seizures plus (GEFS+), infantile focal migrating partial seizures, Lennox-Gastaut syndrome, West syndrome, and seizures.

[0148] In particular, with regard to Dravet syndrome, 80% of patients with Dravet syndrome test positive for SCN1A mutations, but the absence of SCN1A mutations does not rule out the diagnosis of Dravet syndrome. Dravet syndrome is associated with mutations in SCN1A (e.g., partial or whole deletion mutations, truncating mutations, and / or missense mutations in the S4-S6 potential or pore regions), SCN1B (encoding the sodium channel β1 subunit), SCN2A, SCN3A, SCN9A, GABRG2 (encoding the γ2 subunit of the GABA receptor), GABRD (encoding the δ subunit of the GABA receptor), and / or PCDH19.

[0149] The method described herein may be particularly useful for treating children and infants, and may be particularly useful for treating disorders that develop in infancy or childhood.In certain embodiments, the patient of the method of the present disclosure is a newborn, an infant, a toddler, a preschooler, a child of school age, or a young adult.In certain embodiments, the patient is 18 years old or younger, 12 years old or younger, 10 years old or younger, 8 years old or younger, 6 years old or younger, 4 years old or younger, 2 years old or younger, or 1 year old or younger.In certain embodiments, the patient is an adult over 18 years old.

[0150] In certain embodiments, the methods of the present disclosure reduce or prevent seizures or their symptoms in patients in need thereof. In certain embodiments, the methods provided herein may reduce or prevent one or more types of seizures. Ideally, the methods of the present disclosure prevent seizures altogether. However, the present disclosure further includes methods that reduce the number of seizures by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0151] In general, seizures may include convulsions, repetitive movements, abnormal sensations, and combinations of these. Seizures can be classified as focal seizures (also called partial seizures) and generalized seizures. Focal seizures affect only one side of the brain, while generalized seizures affect both sides of the brain. Specific types of focal seizures include simple focal seizures, complex focal seizures, and secondarily generalized seizures. Simple focal seizures may be limited to or concentrated in a particular brain lobe (e.g., the temporal, frontal, parietal, or occipital lobe). Complex focal seizures typically affect a wider area of ​​the brain hemisphere than simple focal seizures, but commonly originate in the temporal or frontal lobe. When a focal seizure spreads from one side of the brain (hemisphere) to both sides, the seizure is called secondarily generalized. Specific types of generalized seizures include absence (also called petit mal), tonic, atonic, myoclonic, tonic-clonic (also called grand mal), and clonic seizures.

[0152] In certain embodiments, the methods described herein reduce the frequency of seizures, reduce the severity of seizures, change the type of seizures (e.g., change the type of seizures from a more severe type to a less severe type), or achieve a combination thereof in a patient after treatment compared to the absence of treatment (e.g., before treatment) or compared to treatment with another conventional therapy.

[0153] The compositions of the present disclosure can be administered via any suitable route. For example, in certain embodiments, administration can include administration to cells or tissue slices for research purposes related to the dysfunction of Nav1.1 sodium channels. In certain embodiments, administration to cells or tissues can be carried out by pipette or injection.

[0154] In certain embodiments, the administration is to a subject and may be intravenous, retro-orbital, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular (ICV), intramuscular, intraparenchymal, intrathecal, intraspinal, intraperitoneal, oral, intranasal, or directly to a targeted site. Delivery may be by needle or cannula or other techniques that expel fluid materials.

[0155] Methods of administration may include those described in US Pat. No. 5,543,158; US Pat. No. 5,641,515 and US Pat. No. 5,399,363.

[0156] As is well known in the medical field, the dose administered to a subject will depend on a variety of factors, such as the subject's body size, surface area, and age, the particular compound administered, sex, duration and route of administration, general health, and other concomitant medications. While doses of compounds of the present disclosure may vary, in certain embodiments, the dose of an artificial expression construct of the present disclosure is 10 5 ~10 100In certain embodiments, patients receiving intravenous, intraparenchymal, intraspinal, retroorbital, or intrathecal administration may receive 10 copies of the 6 ~10 22 A copy of the artificial expression construct can be injected.

[0157] A therapeutically effective amount includes an effective amount and / or an amount capable of providing therapeutic treatment.

[0158] An "effective amount" is the amount of a composition required to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amount disclosed herein is an amount that can produce a statistically significant effect in an animal model, human study, in vivo assay, or in vitro assay related to a disorder associated with dysfunction of the Nav1.1 sodium channel.

[0159] "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of a disorder associated with dysfunction of the Nav1.1 sodium channel, and is administered to a subject with the intent of reducing or eliminating the signs or symptoms of the disorder associated with dysfunction of the Nav1.1 sodium channel. Therapeutic treatment may inhibit, control, or eliminate the presence or activity of the disorder or the cause of the disorder, and / or may inhibit, control, or eliminate the side effects of the disorder.

[0160] The amount of expression construct and the duration of administration of such compositions will be determined by one of skill in the art having the benefit of the teachings of the present invention. However, it is contemplated that administration of an effective amount of the compositions of the present disclosure may be accomplished by a single administration, e.g., by a single injection of a sufficient number of expression constructs to provide a therapeutic benefit to the subject receiving the administration. Alternatively, in some situations, multiple or sequential administrations of an artificial expression construct composition or other genetic construct over a relatively short or longer period of time may be desirable, and the decision to administer such administration may be determined by the person overseeing the administration of such compositions.

[0161] For example, the number of expression constructs administered to a subject may be in the range of 10 to 100, as the dose that may be required to achieve a desired physiological result. 7 pieces / ml, 10 8 pieces / ml, 10 9 pieces / ml, 10 10 pieces / ml, 10 11 pieces / ml, 10 12 pieces / ml, 10 13 The amount of each expression construct may be in the range of 1000 to 15000 cells / ml or more, and these doses may be administered as a single dose or as two or more divided doses. In certain embodiments, it may be desirable to administer two or more expression constructs alone or in combination with one or more additional therapeutic agents to achieve the desired effect of a particular treatment regimen. In certain embodiments, it may be desirable to administer two or more expression constructs in combination to achieve the desired effect.

[0162] The particular dosage and timing of administration for a particular subject can be selected by the treating physician, researcher, or veterinarian, i.e., the amount and time of administration of the composition and / or expression construct will be determined by one of ordinary skill in the art having the benefit of the teachings of the present invention.

[0163] In certain embodiments, treatment of Nav1.1 sodium channelopathy can be combined with other treatments. For example, conventional common treatments for seizures and epilepsy include antiepileptic and non-antiepileptic drug treatments, such as low-carbohydrate diets (e.g., ketogenic diets, e.g., classic ketogenic diets, medium-chain triglyceride (MCT) diets, modified Atkins diets (MAD) and low glycemic index treatments (LGIT)), intravenous immunoglobulin, steroids, elimination diets, vagus nerve stimulation, corticotomy, and subpial polytomy.

[0164] Common antiepileptic and anticonvulsant active compounds that may be used in combination with the compositions described herein include acetazolamide, cannabidiol, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, pregabalin, primidone, retigabine, rufinamide, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, and zonisamide.

[0165] In certain embodiments, a method for rescuing voltage-gated sodium channel function in a target cell population comprises co-administering a therapeutically effective amount of a first artificial expression construct described elsewhere herein and a therapeutically effective amount of a second artificial expression construct to a sample or subject comprising the target cell population, thereby rescuing voltage-gated sodium channel function in the target cell population. The term "co-administration" refers to administering two or more different constructs in a manner such that their expression products interact intracellularly to form functional SCN1A. This co-administration may be simultaneous or may include sequential administration.

[0166] (vi) Kits and commercial packages Kits and commercial packages include the artificial expression constructs described herein. The artificial expression constructs can be isolated. In certain embodiments, the components of the expression product can be separated from each other. In certain embodiments, the expression product is found in a vector, a viral vector, a cell, a tissue section or tissue sample, and / or a transgenic animal. In certain embodiments, the animal is an animal transformed (transgenic animal) by administering a composition comprising an expression construct of the present disclosure. In certain embodiments, the transgenic animal contains genetic modifications that make the animal suitable for use as an animal model for Dravet syndrome. For example, transgenic animals such as mice express Scn1a+ / - may be.

[0167] Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or means for delivering the compositions of the invention (e.g., syringes, injections, etc.).

[0168] Embodiments of the kit or commercial package further include instructions for use of the components included in the kit or commercial package, for example, in basic research, electrophysiological studies, neuroanatomical studies, and / or in the study and / or treatment of disorders, diseases or conditions (e.g., Nav1.1 sodium channel dysfunction such as epilepsy and / or Dravet syndrome).

[0169] The following illustrative embodiments and experimental examples are provided to illustrate specific embodiments of the present disclosure. Those skilled in the art, having reference to this disclosure, will appreciate that various modifications can be made to the specific embodiments disclosed herein while still obtaining the same or similar results without departing from the spirit and scope of the present disclosure.

[0170] (vii) Exemplary Embodiments 1. A system for expressing a coding sequence for SCN1A in a subject in need thereof, comprising: (i) a first artificial expression construct; (ii) a second artificial expression construct; and Including, a first artificial expression construct comprising a first portion of the SCN1A coding sequence, an N-intein coding sequence, and a first promoter sequence, wherein the N-intein coding sequence is located at the 3' end of the first portion of the SCN1A coding sequence; a second artificial expression construct comprising a second portion of the SCN1A coding sequence, a C-intein coding sequence, and a second promoter sequence, wherein the C-intein coding sequence is located at the 5' end of the second portion of the SCN1A coding sequence; A system in which, when the first artificial expression construct and the second artificial expression construct are expressed in a central nervous system cell, the protein product of the first part of the SCN1A coding sequence and the protein product of the second part of the SCN1A coding sequence are linked in the central nervous system cell to form a functional SCN1A protein. 2. The system described in embodiment 1, wherein the first portion of the coding sequence of SCN1A comprises a sequence having at least 90% sequence identity to SEQ ID NO: 59, SEQ ID NO: 61 or SEQ ID NO: 63. 3. The system of embodiment 1 or 2, wherein the first portion of the SCN1A coding sequence comprises hSCN1A-CO-Nterm1049 (sequence number 59), hSCN1A-CO-Nterm956 (sequence number 61), or hSCN1A-CO-Nterm947 (sequence number 63). 4. A system described in any one of embodiments 1 to 3, wherein the second portion of the SCN1A coding sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO: 64. 5. The system of any one of embodiments 1 to 4, wherein the second portion of the SCN1A coding sequence comprises hSCN1A-CO-Cterm949 (sequence number 60), hSCN1A-CO-Cterm1042 (sequence number 62), or hSCN1A-CO-Cterm1051 (sequence number 64). 6. The system of any one of embodiments 1 to 5, wherein the protein product of the coding sequence of the N-intein and the protein product of the coding sequence of the C-intein comprise a split intein after protein splicing. 7. A system described in any one of embodiments 1 to 6, wherein the coding sequence of the N-intein comprises the coding sequence of Cfa-N. 8. The system described in embodiment 7, wherein the coding sequence of Cfa-N comprises the sequence shown in SEQ ID NO: 57 or a sequence having at least 90% sequence identity with SEQ ID NO: 57. 9. A system described in any one of embodiments 1 to 8, wherein the coding sequence of the C-intein comprises the coding sequence of Cfa-C. 10. The system described in embodiment 9, wherein the coding sequence of Cfa-C comprises the sequence shown in SEQ ID NO: 58 or a sequence having at least 90% sequence identity with SEQ ID NO: 58. 11. A system described in any one of embodiments 1 to 10, wherein the first promoter comprises a minBglobin promoter, an hSyn1 promoter, a truncated hSyn1 promoter, or a CMV promoter. 12. The system described in embodiment 11, wherein the minBglobin promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 90% sequence identity with SEQ ID NO: 3. 13. The system described in embodiment 11, wherein the hSyn1 promoter comprises the sequence shown in SEQ ID NO: 52 or a sequence having at least 90% sequence identity with SEQ ID NO: 52. 14. The system described in embodiment 11, wherein the truncated hSyn1 promoter comprises the sequence shown in SEQ ID NO: 54 or a sequence having at least 90% sequence identity with SEQ ID NO: 54. 15. The system described in embodiment 11, wherein the CMV promoter comprises the sequence shown in SEQ ID NO: 4 or a sequence having at least 90% sequence identity with SEQ ID NO: 4. 16. A system described in any one of embodiments 1 to 15, wherein the second promoter comprises a minBglobin promoter, an hSyn1 promoter, a truncated hSyn1 promoter, or a CMV promoter. 17. The system described in embodiment 16, wherein the minBglobin promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 90% sequence identity with SEQ ID NO: 3. 18. The system described in embodiment 16, wherein the hSyn1 promoter comprises the sequence shown in SEQ ID NO: 52 or a sequence having at least 90% sequence identity with SEQ ID NO: 52. 19. The system described in embodiment 16, wherein the truncated hSyn1 promoter comprises the sequence shown in SEQ ID NO: 54 or a sequence having at least 90% sequence identity with SEQ ID NO: 54. 20. The system described in embodiment 16, wherein the CMV promoter comprises the sequence shown in SEQ ID NO: 4 or a sequence having at least 90% sequence identity with SEQ ID NO: 4. 21. A system described in any one of embodiments 1 to 20, wherein the first artificial expression construct further comprises a first enhancer sequence that induces targeted expression of a first portion of the SCN1A coding sequence in a targeted type of central nervous system cell. 22. A system described in any one of embodiments 1 to 21, wherein the second artificial expression construct further comprises a second enhancer sequence that induces targeted expression of a second portion of the SCN1A coding sequence in a targeted type of central nervous system cell. 23. A system described in any one of embodiments 1 to 22, wherein the first artificial expression construct further comprises a first enhancer sequence that induces targeted expression of a first portion of the SCN1A coding sequence in a central nervous system cell of a target type, and the second artificial expression construct further comprises a second enhancer sequence that induces targeted expression of a second portion of the SCN1A coding sequence in a central nervous system cell of the target type. 24. A system described in any one of embodiments 1 to 23, wherein the targeted type of central nervous system cell comprises a neuron. 25. The system of embodiment 24, wherein the neurons include GABAergic neurons or glutamatergic neurons. 26. The system described in embodiment 25, wherein the GABAergic neurons include GABAergic neurons of the forebrain. 27. The system described in embodiment 25 or 26, wherein the GABAergic neurons include entire GABAergic neurons. 28. A system described in any one of embodiments 25 to 27, wherein the glutamatergic neurons include glutamatergic neurons of the forebrain. 29. A system described in any one of embodiments 1 to 28, wherein the targeted type of central nervous system cell comprises a cell line. 30. A system described in any one of embodiments 23 to 29, wherein the first enhancer sequence comprises DLX2.0 (sequence number 2), the second enhancer sequence comprises DLX2.0 (sequence number 2), and the targeted type of central nervous system cell is a GABAergic neuron of the forebrain. 31. A system described in any one of embodiments 23 to 29, wherein the first enhancer sequence comprises eHGT_078h (sequence number 55), the second enhancer sequence comprises eHGT_078h (sequence number 55), and the targeted type of central nervous system cell is a glutamatergic neuron of the forebrain. 32. A system described in any one of embodiments 1 to 31, wherein the first artificial expression construct further comprises a first miRNA binding site that induces targeted expression of a first portion of the SCN1A coding sequence in a targeted type of central nervous system cell. 33. A system described in any one of embodiments 1 to 32, wherein the second artificial expression construct further comprises a second miRNA binding site that induces targeted expression of a second portion of the SCN1A coding sequence in the targeted type of central nervous system cell. 34. A system described in any one of embodiments 1 to 33, wherein the first artificial expression construct further comprises a first miRNA binding site that induces targeted expression of a first portion of the SCN1A coding sequence in a central nervous system cell of the target type, and the second artificial expression construct further comprises a second miRNA binding site that induces targeted expression of a second portion of the SCN1A coding sequence in the central nervous system cell of the target type. 35. The system described in embodiment 34, wherein the first miRNA binding site comprises a 4x2C miRNA binding site (sequence number 56), the second miRNA binding site comprises a 4x2C miRNA binding site (sequence number 56), and the targeted type of central nervous system cell comprises whole GABAergic neurons. 36. A first artificial expression construct and a second artificial expression construct are DLX2.0-minBG-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-SV40pA, DLX2.0-minBG-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-SV40pA; hSyn1-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-shortPolyA; CMV-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN_IRES2_SYFP2_BGHpA, CMV-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG_IRES2_mScarlet-3xNLS_BGHpA; hSyn1short-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-4x2C-WPRE3-shortPolyA, hSyn1short-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-4x2C-WPRE3-shortPolyA; eHGT_078h-minBG-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-SV40pA and eHGT_078h-minBG-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-SV40pA; hSyn1-2xHA-hSCN1A-CO-Nterm956-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm1042-intron-3xFLAG-WPRE3-shortPolyA; hSyn1-2xHA-hSCN1A-CO-Nterm947-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm1051-intron-3xFLAG-WPRE3-shortPolyA; DLX2.0-minBG-hSCN1A-CO-Nterm1049-intron-CfaN-[post-transcriptional regulatory element] DLX2.0-minBG-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; hSyn1-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; CMV-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element]; CMV-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; hSyn1short-hSCN1A-CO-Nterm1049-intron-CfaN-4x2C-[posttranscriptional regulatory element]; hSyn1short-CfaC-hSCN1A-CO-Cterm949-intron-4x2C-[posttranscriptional regulatory element]; eHGT_078h-minBG-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element] eHGT_078h-minBG-CfaC-hSCN1A-CO-Cterm949-intron-[post-transcriptional regulatory element]; hSyn1-hSCN1A-CO-Nterm956-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm1042-intron-[post-transcriptional regulatory element]; and hSyn1-hSCN1A-CO-Nterm947-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm1051-intron-[post-transcriptional regulatory element] A system according to any one of embodiments 1 to 35, each comprising or encoding a set of features selected from: 37. An artificial expression construct comprising a portion of the coding sequence of SCN1A, a coding sequence for an N-intein, and a promoter sequence, wherein the coding sequence for the N-intein is located at the 3' end of a first portion of the coding sequence of SCN1A. 38. An artificial expression construct described in embodiment 37, wherein the portion of the coding sequence of SCN1A comprises a sequence having at least 90% sequence identity to SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63. 39. An artificial expression construct according to embodiment 37 or 38, wherein the portion of the SCN1A coding sequence comprises hSCN1A-CO-Nterm1049 (SEQ ID NO: 59), hSCN1A-CO-Nterm956 (SEQ ID NO: 61), or hSCN1A-CO-Nterm947 (SEQ ID NO: 63). 40. An artificial expression construct described in any one of embodiments 37 to 39, wherein the coding sequence of the N-intein comprises the coding sequence of Cfa-N. 41. An artificial expression construct described in embodiment 40, wherein the coding sequence of Cfa-N comprises the sequence shown in SEQ ID NO: 57 or a sequence having at least 90% sequence identity with SEQ ID NO: 57. 42. An artificial expression construct described in any one of embodiments 37 to 41, wherein the promoter sequence comprises a minBglobin promoter, an hSyn1 promoter, a CMV promoter, or a truncated hSyn1 promoter. 43. An artificial expression construct described in any one of embodiments 37 to 42, further comprising an enhancer sequence that induces targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell. 44. An artificial expression construct described in embodiment 43, wherein the enhancer sequence comprises DLX2.0 (sequence number 2) and the targeted type of central nervous system cell comprises forebrain GABAergic neurons. 45. The artificial expression construct of embodiment 43, wherein the enhancer sequence comprises eHGT_078h and the targeted type of central nervous system cell comprises glutamatergic neurons of the forebrain. 46. ​​An artificial expression construct described in any one of embodiments 37 to 45, further comprising an miRNA binding site that induces targeted expression of a portion of the coding sequence of SCN1A in a targeted type of central nervous system cell. 47. An artificial expression construct described in embodiment 46, wherein the miRNA binding site comprises a 4x2c miRNA binding site and the targeted type of central nervous system cell comprises all GABAergic neurons. 48. An artificial expression construct according to any one of embodiments 37 to 47, further encoding a reporter protein. 49. The artificial expression construct of embodiment 48, wherein the reporter protein comprises a fluorescent protein or a tag cassette. 50. An artificial expression construct described in embodiment 49, wherein the tag cassette comprises the sequences shown in SEQ ID NO: 15 and SEQ ID NOs: 34 to 45. 51. An artificial expression construct described in any one of embodiments 37 to 50, contained within an adeno-associated virus (AAV) vector. 52. An artificial expression construct described in any one of embodiments 37 to 51, which is associated with a capsid that crosses the blood-brain barrier. 53. An artificial expression construct described in embodiment 52, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 54. An artificial expression construct according to any one of embodiments 37 to 53, comprising or encoding a skipping element. 55. An artificial expression construct described in embodiment 54, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 56. The artificial expression construct of embodiment 55, wherein the IRES comprises IRES2. 57. An artificial expression construct described in embodiment 55, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 58. DLX2.0 (SEQ ID NO: 2), 4x2C miR binding site (SEQ ID NO: 56), 8x2C 58. The artificial expression construct of any one of embodiments 37 to 57, comprising or encoding a set of features selected from: miR binding site (SEQ ID NO: 87), eHGT_078h, intein, AAV, scAAV, rAAV, pAAV, pSMART-HCKan, minBglobin, CMV promoter, hSyn1 promoter, truncated hSyn1 promoter, minCMV, minCMV*, minRho, minRho*, fluorescent protein (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, intron, 10aa, nuclear export protein, self-cleaving peptide, WPRE, WPRE3, hGHpA, SV40pA, shortPolyA and / or BGHpA. 59. An artificial expression construct comprising a portion of the coding sequence of SCN1A, a coding sequence of a C-intein, an enhancer sequence that induces targeted expression of the portion of the coding sequence of SCN1A in a target type of central nervous system cell, and a promoter sequence, wherein the coding sequence of the C-intein is located 5' to the portion of the coding sequence of SCN1A. 60. An artificial expression construct described in embodiment 59, wherein the second portion of the SCN1A coding sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64. 61. The artificial expression construct of embodiment 59 or 60, wherein the second part of the SCN1A coding sequence comprises hSCN1A-CO-Cterm949 (SEQ ID NO: 60), hSCN1A-CO-Cterm1042 (SEQ ID NO: 62), or hSCN1A-CO-Cterm1051 (SEQ ID NO: 64). 62. An artificial expression construct described in any one of embodiments 59 to 61, wherein the coding sequence of the C-intein comprises the coding sequence of Cfa-C. 63. An artificial expression construct described in embodiment 62, wherein the coding sequence of Cfa-C comprises the sequence shown in SEQ ID NO: 58 or a sequence having at least 90% sequence identity with SEQ ID NO: 58. 64. An artificial expression construct described in any one of embodiments 59 to 63, further comprising an enhancer sequence that induces targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell. 65. An artificial expression construct described in embodiment 64, wherein the enhancer sequence comprises DLX2.0 (sequence number 2) and the targeted type of central nervous system cell comprises forebrain GABAergic neurons. 66. The artificial expression construct of embodiment 64, wherein the enhancer sequence comprises eHGT_078h and the targeted type of central nervous system cell comprises glutamatergic neurons of the forebrain. 67. An artificial expression construct described in any one of embodiments 59 to 66, further comprising an miRNA binding site that induces targeted expression of a portion of the coding sequence of SCN1A in a targeted type of central nervous system cell. 68. An artificial expression construct described in embodiment 67, wherein the miRNA binding site comprises a 4x2c miRNA binding site and the targeted type of central nervous system cell comprises whole GABAergic neurons. 69. An artificial expression construct described in any one of embodiments 59 to 68, further encoding a reporter protein. 70. The artificial expression construct described in embodiment 69, wherein the reporter protein comprises a fluorescent protein or a tag cassette. 71. An artificial expression construct described in embodiment 70, wherein the tag cassette comprises the sequences shown in SEQ ID NO: 15 and SEQ ID NOs: 34 to 45. 72. An artificial expression construct described in any one of embodiments 59 to 71, contained within an adeno-associated virus (AAV) vector. 73. An artificial expression construct described in any one of embodiments 59 to 72, which is associated with a capsid that crosses the blood-brain barrier. 74. The artificial expression construct of embodiment 73, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, or AAV-PPS. 75. An artificial expression construct according to any one of embodiments 59 to 74, comprising or encoding a skipping element. 76. The artificial expression construct of embodiment 75, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES). 77. The artificial expression construct of embodiment 76, wherein the IRES comprises IRES2. 78. An artificial expression construct described in embodiment 76 or 77, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A. 79. DLX2.0 (SEQ ID NO: 2), 4x2C miR binding site (SEQ ID NO: 56), 8x2C 79. The artificial expression construct of any one of embodiments 59 to 78, comprising or encoding a set of features selected from: miR binding site (SEQ ID NO: 87), eHGT_078h, intein, AAV, scAAV, rAAV, pAAV, pSMART-HCKan, minBglobin, CMV promoter, hSyn1 promoter, truncated hSyn1 promoter, minCMV, minCMV*, minRho, minRho*, fluorescent protein (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), tag cassette, intron, 10aa, nuclear export protein, self-cleaving peptide, WPRE, WPRE3, hGHpA, SV40pA, shortPolyA and / or BGHpA. 80. An administrable composition comprising a system described in any one of embodiments 1 to 36, an artificial expression construct described in any one of embodiments 37 to 58, and / or an artificial expression construct described in any one of embodiments 59 to 79. 81. A vector comprising an artificial expression construct according to any one of embodiments 37 to 58 or an artificial expression construct according to any one of embodiments 59 to 79. 82. The vector described in embodiment 81, which is a viral vector. 83. A vector system, comprising the system of any one of embodiments 1 to 36, wherein the first artificial expression construct is contained within the first vector and the second artificial expression construct is contained within the second vector. 84. The vector system of embodiment 83, wherein the first vector comprises components of CN3252, CN3683, CN3251, CN3677, CN4541, CN4217, or CN4642. 85. The vector system of embodiment 83 or 84, wherein the second vector comprises components of CN3254, CN3684, CN3253, CN3678, CN4542, CN4218, or CN4643. 86. A transgenic cell comprising a system described in any one of embodiments 1 to 46. 87. The transgenic cell of embodiment 86, comprising a neuron. 88. The transgenic cell of embodiment 86 or 87, which is a GABAergic neuron or a glutamatergic neuron. 89. The transgenic cell described in embodiment 88, wherein the GABAergic neurons are whole GABAergic neurons, GABAergic neurons of the forebrain, GABAergic neurons of the hippocampus, and / or GABAergic neurons of the cortex. 90. The transgenic cell described in embodiment 88 or 89, wherein the GABAergic neuron is a GABAergic neuron of the forebrain. 91. The transgenic cell of embodiment 88 or 89, wherein the glutamatergic neurons include glutamatergic neurons of the forebrain. 92. A transgenic cell described in any one of embodiments 86 to 91, including a cell line. 93. A transgenic cell described in any one of embodiments 86 to 92, which is a mouse cell, a human cell, or a non-human primate cell. 94. A non-human transgenic animal comprising a system described in any one of embodiments 1 to 36 and / or a transgenic cell described in any one of embodiments 86 to 93. 95. The non-human transgenic animal of embodiment 94, which is a mouse or a non-human primate. 96. A kit comprising a system according to any one of embodiments 1 to 36, an artificial expression construct according to any one of embodiments 37 to 58, an artificial expression construct according to any one of embodiments 58 to 79, a transgenic cell according to any one of embodiments 86 to 93, and / or a non-human transgenic animal according to embodiment 94 or 95. 97. A method for rescuing voltage-gated sodium channel function in a targeted cell population, comprising co-administering a therapeutically effective amount of an artificial expression construct described in any one of embodiments 37-58 and a therapeutically effective amount of an artificial expression construct described in any one of embodiments 59-79 to a sample or subject comprising the targeted cell population, thereby rescuing voltage-gated sodium channel function in the targeted cell population. 98. The method of embodiment 97, wherein the co-administration comprises pipetting. 99. The method described in embodiment 98, wherein the pipetting is performed on a brain slice. 100. The method of embodiment 99, wherein the brain slice comprises neurons. 101. The method of embodiment 99 or 100, wherein the brain slice comprises GABAergic neurons or glutamatergic neurons. 102. The method of embodiment 101, wherein the GABAergic neurons include whole GABAergic neurons, GABAergic neurons of the forebrain, GABAergic neurons of the hippocampus, or GABAergic neurons of the cortex. 103. The method of embodiment 101 or 102, wherein the glutamatergic neurons comprise glutamatergic neurons of the forebrain. 104. The method of any one of embodiments 99 to 103, wherein the brain slice comprises a cell line. 105. The method of any one of embodiments 99 to 104, wherein the brain slice is a mouse, human, or non-human primate brain slice. 106. The method of any one of embodiments 97 to 105, wherein said co-administration comprises administration to a living subject. 107. The method of embodiment 106, wherein the living subject is a human, a non-human primate, or a mouse. 108. The method of embodiment 106 or 107, wherein the living subject is a subject in need of rescue of voltage-gated sodium channel function because the subject has been diagnosed with an SCN1A-associated seizure disorder. 109. The method of embodiment 108, wherein the SCN1A-associated seizure disorder comprises Dravet syndrome, myoclonic seizures, myoclonic-astatic epilepsy (MAE), refractory childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epileptic febrile seizures plus (GEFS+), infantile focal migrating partial seizures, Lennox-Gastaut syndrome, or West syndrome. 110. The method of any one of embodiments 106 to 109, wherein the living subject is a pediatric patient. 111. The method of any one of embodiments 106 to 110, wherein the living subject is under 4 years of age. 112. The method of any one of embodiments 106 to 109, wherein the living subject is a non-human transgenic animal. 113. The method of any one of embodiments 97 to 112, wherein the co-administration comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection. 114. An artificial expression construct comprising CN3252, CN3254, CN3683, CN3684, CN3251, CN3253, CN3677, CN3678, CN4541, CN4542, CN4217, CN4218, CN4642, or CN4643.

[0171] (viii) Conclusion The nucleic acid and amino acid sequences provided herein are represented by the abbreviations used for nucleotide bases and amino acid residues as set forth in 37 CFR §§ 1.831-1.835 and as set forth in WIPO Standard ST.26 (effective July 1, 2022). Although only one strand is shown for each nucleic acid sequence, the complementary strand, if applicable, is also included in the embodiments.

[0172] Variants of the sequences disclosed and cited herein are also encompassed by this application. Guidelines for determining which amino acid residues can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNASTAR. TM Software (Madison, Wisconsin, USA) can be used to find amino acid changes. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of amino acids with similarly charged amino acids or with uncharged amino acids. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0173] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can usually be made without altering the biological activity of the resulting molecule. Those skilled in the art will be familiar with the fact that substituting a single amino acid in a non-critical region of a polypeptide usually does not substantially alter the biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are typically classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2 (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3 (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5 (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (Ile), leucine (Leu), and arginine (Arg). Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic nonpolar or slightly polar aliphatic residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0174] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0175] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity index will result in a protein with similar biological activity, i.e., a protein with equivalent biological functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0176] As detailed in U.S. Patent No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that certain amino acids can be substituted with other amino acids having similar hydrophilicity values, and that such substitutions can result in biologically equivalent, and particularly immunologically equivalent, proteins. When making such changes, substitutions between amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions between amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values ​​within ±0.5 are even more particularly preferred.

[0177] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0178] As described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0179] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences that have at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to a protein, nucleic acid or gene sequence disclosed herein.

[0180] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid, or gene sequences, as determined by the matching between strings of protein, nucleic acid, or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to obtain the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and identity calculations may be performed using the Megalign program within the LASERGENE suite of bioinformatics computing software (DNASTAR, Madison, Wis.).Multiple sequence alignment can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Further relevant programs include the GCG program suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, pp. 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that are the basis of the program. In this specification, "default values" refers to a set of numerical values ​​or parameters that are pre-registered in the software when the software is initialized.

[0181] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20µg / ml denatured fragment-treated salmon sperm DNA, followed by washing the filter with 0.1xSSC at 50°C. The stringency of hybridization and signal detection can be changed mainly by adjusting the concentration of formamide (lowering the percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37°C in a solution containing 6x SSPE (20x SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml blocking salmon sperm DNA, followed by a wash at 50°C with 1x SSPE and 0.1% SDS. Even lower stringency can be achieved by performing stringent post-hybridization washes at a high salt concentration (e.g., 5x SSC). These conditions can be varied by adding and / or substituting alternative blocking reagents used to reduce background in hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0182] The term "concatemerize" is used in a broad sense to mean to link in a chain or to link in a series. This term is used to describe the linking of multiple nucleotide sequences to obtain a single nucleotide sequence or the linking of multiple amino acid sequences to obtain a single amino acid sequence. Also, "concatemerize" is understood to refer to "concatenation."

[0183] As will be understood by those skilled in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the specific components, steps, materials, or ingredients described. Accordingly, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprising" means the inclusion of, but is not limited to, any unrecited component, step, material, or ingredient, even if in greater amounts. The transitional phrase "consisting of" excludes any unrecited component, step, material, or ingredient. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the recited components, steps, materials, or ingredients, as well as components, steps, materials, or ingredients that do not materially affect the embodiment. A significant effect is a statistically significant reduction in target expression of SCN1A in a targeted cell type using the artificial expression constructs disclosed herein.

[0184] In certain embodiments, "artificial" means not naturally occurring.

[0185] Unless otherwise indicated, all numerical values ​​in the specification and claims expressing quantities or properties of materials, such as molecular weight or reaction conditions, are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. More specifically, the term "about," when used in conjunction with a stated value or range, has the meaning reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0186] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0187] As used in describing the present invention (particularly in the claims that follow), the terms "a," "an," "the," and similar designators are intended to encompass both the singular and the plural unless otherwise indicated or the context clearly dictates otherwise. Numerical ranges recited herein are intended as a shorthand way of referring individually to each numerical value within the range. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any methods described herein can be performed in any suitable order unless otherwise indicated or the context clearly dictates otherwise. The use of any examples or illustrative language (e.g., "etc.") provided herein is for the purpose of illustrating the invention only and does not limit the scope of the invention as recited in the claims. No term used herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0188] Groupings of other elements of the invention or of various embodiments of the invention disclosed herein should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that one or more members of a group may be added to another group, or one or more members may be deleted from a group, for reasons of convenience and / or patentability. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0189] Specific embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, those skilled in the art will readily recognize from a review of the foregoing detailed description that the embodiments described herein can be modified in various ways. The inventors anticipate that such modifications may be accommodated by those skilled in the art, and intend that the invention may be practiced in ways other than those specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter to the fullest extent permitted by applicable law. Furthermore, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless expressly stated otherwise or the context clearly dictates otherwise.

[0190] Additionally, throughout this specification, various patents, publications, journal articles, and other documents are cited (the "references"). Each reference cited herein is individually incorporated herein by reference for the teachings thereof.

[0191] Finally, the embodiments of the invention disclosed herein are to be considered illustrative of the principles of the invention. Other modifications may be employed within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Therefore, the invention is not to be limited to what is precisely as shown and described herein.

[0192] The details set forth herein are by way of example only and are intended to illustrate preferred embodiments of the invention, and are presented to provide what is believed to be the most useful and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the invention. In this regard, no structural details of the invention have been described in more detail than is necessary for a fundamental understanding of the invention, and those skilled in the art will be able to readily understand how to actually embody several forms of the invention by perusing the description of the invention with reference to the drawings and / or examples.

[0193] The definitions and explanations used in this disclosure are intended to control future interpretations unless clearly and unambiguously changed in the examples below, or unless the interpretation becomes meaningless or substantially meaningless due to the meaning of the terms. If the definition of a term becomes meaningless or substantially meaningless due to the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. A system for expressing a functional SCN1A protein in a subject in need thereof, comprising: (i) a first artificial expression construct; (ii) a second artificial expression construct; and Including, a first artificial expression construct comprising: a first portion of the coding sequence of SCN1A having the sequence set forth in SEQ ID NO:59; The coding sequence of the N-intein shown in SEQ ID NO: 57; a first promoter sequence; and Including, the coding sequence for the N-intein is located at the 3' end of the first portion of the coding sequence for SCN1A; a second artificial expression construct comprising: a second portion of the coding sequence of SCN1A having the sequence set forth in SEQ ID NO: 60; and The coding sequence of the C-intein shown in SEQ ID NO: 58; a second promoter sequence; and Including, the coding sequence for the C-intein is located at the 5' end of the second portion of the coding sequence for SCN1A; A system in which, when the first artificial expression construct and the second artificial expression construct are expressed in a central nervous system cell, the protein product of the first part of the SCN1A coding sequence and the protein product of the second part of the SCN1A coding sequence are linked in the central nervous system cell to form a functional SCN1A protein.

2. A system for expressing a functional SCN1A protein in a subject in need thereof, comprising: (i) a first artificial expression construct; (ii) a second artificial expression construct; and Including, a first artificial expression construct comprising a first portion of the coding sequence of SCN1A, a coding sequence of an N-intein, and a first promoter sequence, wherein the coding sequence of the N-intein is located at the 3' end of the first portion of the coding sequence of SCN1A; a second artificial expression construct comprising a second portion of the coding sequence of SCN1A, a coding sequence of a C-intein, and a second promoter sequence, wherein the coding sequence of the C-intein is located at the 5' end of the second portion of the coding sequence of SCN1A; A system in which, when the first artificial expression construct and the second artificial expression construct are expressed in a central nervous system cell, the protein product of the first part of the SCN1A coding sequence and the protein product of the second part of the SCN1A coding sequence are linked in the central nervous system cell to form a functional SCN1A protein.

3. The system of claim 2, wherein the first portion of the SCN1A coding sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 59, SEQ ID NO: 61 or SEQ ID NO:

63.

4. The system of claim 2, wherein the first portion of the SCN1A coding sequence comprises hSCN1A-CO-Nterm1049 (SEQ ID NO: 59), hSCN1A-CO-Nterm956 (SEQ ID NO: 61), or hSCN1A-CO-Nterm947 (SEQ ID NO: 63).

5. The system of claim 2, wherein the second portion of the SCN1A coding sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO:

64.

6. The system of claim 2, wherein the second portion of the SCN1A coding sequence comprises hSCN1A-CO-Cterm949 (sequence number 60), hSCN1A-CO-Cterm1042 (sequence number 62), or hSCN1A-CO-Cterm1051 (sequence number 64).

7. 3. The system of claim 2, wherein the protein product of the coding sequence for the N-intein and the protein product of the coding sequence for the C-intein comprise a split intein after protein splicing.

8. The system of claim 2, wherein the coding sequence of the N-intein comprises the coding sequence of Cfa-N.

9. The system of claim 8, wherein the coding sequence of Cfa-N comprises the sequence shown in SEQ ID NO: 57 or a sequence having at least 90% sequence identity with SEQ ID NO:

57.

10. The system of claim 2, wherein the coding sequence of the C-intein comprises the coding sequence of Cfa-C.

11. The system of claim 10, wherein the coding sequence of Cfa-C comprises the sequence shown in SEQ ID NO: 58 or a sequence having at least 90% sequence identity with SEQ ID NO:

58.

12. The system of claim 2 , wherein the first promoter comprises a minBglobin promoter, an hSyn1 promoter, a truncated hSyn1 promoter, or a CMV promoter.

13. The system of claim 12, wherein the minBglobin promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 90% sequence identity with SEQ ID NO:

3.

14. The system of claim 12, wherein the hSyn1 promoter comprises the sequence shown in SEQ ID NO: 52 or a sequence having at least 90% sequence identity with SEQ ID NO:

52.

15. The system of claim 12, wherein the truncated hSyn1 promoter comprises the sequence shown in SEQ ID NO: 54 or a sequence having at least 90% sequence identity with SEQ ID NO:

54.

16. The system of claim 12, wherein the CMV promoter comprises the sequence shown in SEQ ID NO:4 or a sequence having at least 90% sequence identity with SEQ ID NO:

4.

17. The system of claim 2 , wherein the second promoter comprises a minBglobin promoter, an hSyn1 promoter, a truncated hSyn1 promoter, or a CMV promoter.

18. The system of claim 12, wherein the minBglobin promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 90% sequence identity with SEQ ID NO:

3.

19. The system of claim 12, wherein the hSyn1 promoter comprises the sequence shown in SEQ ID NO: 52 or a sequence having at least 90% sequence identity with SEQ ID NO:

52.

20. The system of claim 14, wherein the truncated hSyn1 promoter comprises the sequence shown in SEQ ID NO: 54 or a sequence having at least 90% sequence identity with SEQ ID NO:

54.

21. The system of claim 12, wherein the CMV promoter comprises the sequence shown in SEQ ID NO:4 or a sequence having at least 90% sequence identity with SEQ ID NO:

4.

22. 3. The system of claim 2, wherein the first artificial expression construct further comprises a first enhancer sequence that induces targeted expression of a first portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

23. The system of claim 2, wherein the second artificial expression construct further comprises a second enhancer sequence that induces targeted expression of a second portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

24. 3. The system of claim 2, wherein the first artificial expression construct further comprises a first enhancer sequence that induces targeted expression of a first portion of the SCN1A coding sequence in a target type of central nervous system cell, and the second artificial expression construct further comprises a second enhancer sequence that induces targeted expression of a second portion of the SCN1A coding sequence in the target type of central nervous system cell.

25. The system of claim 2 , wherein the targeted type of central nervous system cell comprises a neuron.

26. 26. The system of claim 25, wherein the neurons comprise GABAergic neurons or glutamatergic neurons.

27. 27. The system of claim 26, wherein the GABAergic neurons include GABAergic neurons of the forebrain.

28. 27. The system of claim 26, wherein the GABAergic neurons include entire GABAergic neurons.

29. 27. The system of claim 26, wherein the glutamatergic neurons comprise glutamatergic neurons of the forebrain.

30. The system of claim 2 , wherein the targeted type of central nervous system cell comprises a cell line.

31. The system of claim 24, wherein the first enhancer sequence comprises DLX2.0 (sequence number 2), the second enhancer sequence comprises DLX2.0 (sequence number 2), and the targeted type of central nervous system cell is a GABAergic neuron of the forebrain.

32. 25. The system of claim 24, wherein the first enhancer sequence comprises eHGT_078h (SEQ ID NO: 55), the second enhancer sequence comprises eHGT_078h (SEQ ID NO: 55), and the targeted type of central nervous system cell is a glutamatergic neuron of the forebrain.

33. 3. The system of claim 2, wherein the first artificial expression construct further comprises a first miRNA binding site that induces targeted expression of a first portion of the SCN1A coding sequence in a target type of central nervous system cell.

34. The system of claim 2, wherein the second artificial expression construct further comprises a second miRNA binding site that directs targeted expression of a second portion of the SCN1A coding sequence in a target type of central nervous system cell.

35. 3. The system of claim 2, wherein the first artificial expression construct further comprises a first miRNA binding site that induces targeted expression of a first portion of the SCN1A coding sequence in a target type of central nervous system cell, and the second artificial expression construct further comprises a second miRNA binding site that induces targeted expression of a second portion of the SCN1A coding sequence in the target type of central nervous system cell.

36. The system of claim 35, wherein the first miRNA binding site comprises a 4x2C miRNA binding site (sequence number 56), the second miRNA binding site comprises a 4x2C miRNA binding site (sequence number 56), and the targeted type of central nervous system cell comprises all GABAergic neurons.

37. a first artificial expression construct and a second artificial expression construct, DLX2.0-minBglobin-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-SV40pA, DLX2.0-minBglobin-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-SV40pA; hSyn1-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-shortPolyA; CMV promoter-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN_IRES2_SYFP2_BGHpA, CMV promoter-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG_IRES2_mScarlet-3xNLS_BGHpA; hSyn1short-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-4x2C-WPRE3-shortPolyA, hSyn1short-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-4x2C-WPRE3-shortPolyA; eHGT_078h-minBglobin-2xHA-hSCN1A-CO-Nterm1049-intron-CfaN-WPRE3-SV40pA and eHGT_078h-minBglobin-CfaC-hSCN1A-CO-Cterm949-intron-3xFLAG-WPRE3-SV40pA; hSyn1-2xHA-hSCN1A-CO-Nterm956-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm1042-intron-3xFLAG-WPRE3-shortPolyA; hSyn1-2xHA-hSCN1A-CO-Nterm947-intron-CfaN-WPRE3-shortPolyA, hSyn1-CfaC-hSCN1A-CO-Cterm1051-intron-3xFLAG-WPRE3-shortPolyA; DLX2.0-minBglobin-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element] DLX2.0-minBglobin-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; hSyn1-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; CMV promoter-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element]; CMV promoter-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; hSyn1short-hSCN1A-CO-Nterm1049-intron-CfaN-4x2C-[posttranscriptional regulatory element]; hSyn1short-CfaC-hSCN1A-CO-Cterm949-intron-4x2C-[posttranscriptional regulatory element]; eHGT_078h-minBglobin-hSCN1A-CO-Nterm1049-intron-CfaN-[posttranscriptional regulatory element] eHGT_078h-minBglobin-CfaC-hSCN1A-CO-Cterm949-intron-[posttranscriptional regulatory element]; hSyn1-hSCN1A-CO-Nterm956-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm1042-intron-[post-transcriptional regulatory element]; and hSyn1-hSCN1A-CO-Nterm947-intron-CfaN-[posttranscriptional regulatory element]; hSyn1-CfaC-hSCN1A-CO-Cterm1051-intron-[post-transcriptional regulatory element] The system of claim 2 , each including or encoding a set of features selected from:

38. An artificial expression construct comprising a portion of the coding sequence of SCN1A, a coding sequence of an N-intein, and a promoter sequence, wherein the coding sequence of the N-intein is located at the 3' end of the portion of the coding sequence of SCN1A.

39. 39. The artificial expression construct of claim 38, wherein the portion of the coding sequence of SCN1A comprises a sequence having at least 90% sequence identity to SEQ ID NO:59, SEQ ID NO:61 or SEQ ID NO:

63.

40. 39. The artificial expression construct of claim 38, wherein the portion of the SCN1A coding sequence comprises hSCN1A-CO-Nterm1049 (SEQ ID NO: 59), hSCN1A-CO-Nterm956 (SEQ ID NO: 61), or hSCN1A-CO-Nterm947 (SEQ ID NO: 63).

41. 39. The artificial expression construct of Claim 38, wherein the coding sequence of the N-intein comprises the coding sequence of Cfa-N.

42. 42. The artificial expression construct of claim 41, wherein the coding sequence of Cfa-N comprises the sequence set forth in SEQ ID NO:57 or a sequence having at least 90% sequence identity to SEQ ID NO:

57.

43. 39. The artificial expression construct of claim 38, wherein the promoter sequence comprises a minBglobin promoter, an hSyn1 promoter, a CMV promoter, or a truncated hSyn1 promoter.

44. 39. The artificial expression construct of claim 38, further comprising an enhancer sequence that directs targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

45. 45. The artificial expression construct of claim 44, wherein the enhancer sequence comprises DLX2.0 (SEQ ID NO: 2) and the targeted type of central nervous system cell comprises forebrain GABAergic neurons.

46. 45. The artificial expression construct of claim 44, wherein the enhancer sequence comprises eHGT_078h and the targeted type of central nervous system cell comprises glutamatergic neurons of the forebrain.

47. 39. The artificial expression construct of claim 38, further comprising an miRNA binding site that directs targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

48. 48. The artificial expression construct of Claim 47, wherein the miRNA binding site comprises a 4x2c miRNA binding site and the targeted central nervous system cell type comprises whole GABAergic neurons.

49. 39. The artificial expression construct of claim 38, further encoding a reporter protein.

50. 50. The artificial expression construct of claim 49, wherein the reporter protein comprises a fluorescent protein or a tag cassette.

51. 51. The artificial expression construct of claim 50, wherein the tag cassette comprises the sequences set forth in SEQ ID NO: 15 and SEQ ID NOs: 34-45.

52. 39. The artificial expression construct of claim 38, contained within an adeno-associated virus (AAV) vector.

53. 39. The artificial expression construct of claim 38, wherein the construct is associated with a capsid that crosses the blood-brain barrier.

54. 54. The artificial expression construct of claim 53, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV1R6, AAV1R7, AAV9, or AAVrh.

10.

55. 39. The artificial expression construct of claim 38, comprising or encoding a skipping element.

56. 56. The artificial expression construct of claim 55, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

57. 57. The artificial expression construct of claim 56, wherein the IRES comprises IRES2.

58. 57. The artificial expression construct of Claim 56, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.

59. DLX2.0 (SEQ ID NO: 2), 4x2C miR binding site (SEQ ID NO: 56), 8x2C miR binding site (SEQ ID NO: 87), eHGT_078h, intein, split intein (e.g., Cfa-N, Cfa-C), hSCN1A-CO-Nterm1049, hSCN1A-CO-Cterm949, hSCN1A-CO-Nterm956, hSCN1A-CO-Cterm1042, hSCN1A-CO-Nterm947, hSCN1A-CO-Cterm1051, AAV, scAAV, rAAV, pAAV, pSMART-HCKan, minBglobin, CMV promoter, hSyn1 promoter, truncated hSyn1 promoter, 39. The artificial expression construct of claim 38, comprising or encoding a set of features selected from minCMV, minCMV*, minRho, minRho*, a fluorescent protein (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), a tag cassette (e.g., 3xFLAG), an intron, 10aa, a nuclear export protein, a self-cleaving peptide, WPRE, WPRE3, hGHpA, SV40pA, shortPolyA and / or BGHpA.

60. An artificial expression construct comprising a portion of the coding sequence of SCN1A, a coding sequence of a C-intein, an enhancer sequence that induces targeted expression of the portion of the coding sequence of SCN1A in a target type of central nervous system cell, and a promoter sequence, wherein the coding sequence of the C-intein is located at the 5' end of the portion of the coding sequence of SCN1A.

61. 61. The artificial expression construct of claim 60, wherein the second portion of the coding sequence of SCN1A comprises a sequence having at least 90% sequence identity to SEQ ID NO: 60, SEQ ID NO: 62 or SEQ ID NO:

64.

62. 61. The artificial expression construct of claim 60, wherein the second portion of the SCN1A coding sequence comprises hSCN1A-CO-Cterm949 (SEQ ID NO: 60), hSCN1A-CO-Cterm1042 (SEQ ID NO: 62), or hSCN1A-CO-Cterm1051 (SEQ ID NO: 64).

63. 61. The artificial expression construct of claim 60, wherein the coding sequence of the C-intein comprises the coding sequence of Cfa-C.

64. 64. The artificial expression construct of claim 63, wherein the coding sequence of Cfa-C comprises the sequence set forth in SEQ ID NO:58 or a sequence having at least 90% sequence identity to SEQ ID NO:

58.

65. 61. The artificial expression construct of claim 60, further comprising an enhancer sequence that directs targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

66. 66. The artificial expression construct of claim 65, wherein the enhancer sequence comprises DLX2.0 (SEQ ID NO: 2) and the targeted type of central nervous system cell comprises forebrain GABAergic neurons.

67. 66. The artificial expression construct of claim 65, wherein the enhancer sequence comprises eHGT_078h and the targeted type of central nervous system cell comprises forebrain glutamatergic neurons.

68. 61. The artificial expression construct of claim 60, further comprising an miRNA binding site that directs targeted expression of a portion of the SCN1A coding sequence in a targeted type of central nervous system cell.

69. 69. The artificial expression construct of Claim 68, wherein the miRNA binding site comprises a 4x2c miRNA binding site and the targeted central nervous system cell type comprises whole GABAergic neurons.

70. 61. The artificial expression construct of claim 60, further encoding a reporter protein.

71. 71. The artificial expression construct of Claim 70, wherein the reporter protein comprises a fluorescent protein or a tag cassette.

72. 72. The artificial expression construct of claim 71, wherein the tag cassette comprises the sequences set forth in SEQ ID NO: 15 and SEQ ID NOs: 34-45.

73. 61. The artificial expression construct of claim 60, contained within an adeno-associated virus (AAV) vector.

74. 61. The artificial expression construct of claim 60, wherein the construct is associated with a capsid that crosses the blood-brain barrier.

75. 75. The artificial expression construct of claim 74, wherein the capsid comprises PHP.eB, AAV-BR1, AAV-PHP.S, AAV-PHP.B, AAV-PPS, AAV1R6, AAV1R7, AAV9, or AAVrh.

10.

76. 61. The artificial expression construct of claim 60, comprising or encoding a skipping element.

77. 77. The artificial expression construct of claim 76, wherein the skipping element comprises a 2A peptide and / or an internal ribosome entry site (IRES).

78. 78. The artificial expression construct of claim 77, wherein the IRES comprises IRES2.

79. 78. The artificial expression construct of Claim 77, wherein the 2A peptide comprises T2A, P2A, E2A, or F2A.

80. DLX2.0 (SEQ ID NO: 2), 4x2C miR binding site (SEQ ID NO: 56), 8x2C miR binding site (SEQ ID NO: 87), eHGT_078h, intein, split intein (e.g., Cfa-N, Cfa-C), hSCN1A-CO-Nterm1049, hSCN1A-CO-Cterm949, hSCN1A-CO-Nterm956, hSCN1A-CO-Cterm1042, hSCN1A-CO-Nterm947, hSCN1A-CO-Cterm1051, AAV, scAAV, rAAV, pAAV, pSMART-HCKan, minBglobin, CMV promoter, hSyn1 promoter, truncated hSyn1 promoter, 61. The artificial expression construct of claim 60, comprising or encoding a set of features selected from minCMV, minCMV*, minRho, minRho*, a fluorescent protein (e.g., EGFP, SYFP, GFP), hsA2, Cre, iCre, dgCre, FlpO, tTA2, SP10 (e.g., 3xSP10), a tag cassette (e.g., 3xFLAG), an intron, 10aa, a nuclear export protein, a self-cleaving peptide, WPRE, WPRE3, hGHpA, SV40pA, shortPolyA, and / or BGHpA.

81. 61. An administrable composition comprising the system of claim 2, the artificial expression construct of claim 38, and / or the artificial expression construct of claim 60.

82. 61. A vector comprising the artificial expression construct of claim 38 or the artificial expression construct of claim 60.

83. 83. The vector of claim 82, which is a viral vector.

84. 10. A vector system comprising the system of claim 2, wherein the first artificial expression construct is contained within a first vector and the second artificial expression construct is contained within a second vector.

85. 85. The vector system of claim 84, wherein the first vector comprises components of CN3252, CN3683, CN3251, CN3677, CN4541, CN4217, or CN4642.

86. 85. The vector system of claim 84, wherein the second vector comprises components of CN3254, CN3684, CN3253, CN3678, CN4542, CN4218, or CN4643.

87. A transgenic cell comprising the system of claim 1.

88. 88. The transgenic cell of claim 87, comprising a neuron.

89. 88. The transgenic cell of claim 87, which is a GABAergic neuron or a glutamatergic neuron.

90. 90. The transgenic cell of claim 89, wherein the GABAergic neurons are whole GABAergic neurons, forebrain GABAergic neurons, hippocampal GABAergic neurons, and / or cortical GABAergic neurons.

91. 90. The transgenic cell of claim 89, wherein the GABAergic neuron is a GABAergic neuron of the forebrain.

92. 90. The transgenic cell of claim 89, wherein the glutamatergic neurons comprise glutamatergic neurons of the forebrain.

93. 88. The transgenic cell of claim 87, comprising a cell line.

94. 88. The transgenic cell of claim 87, which is a mouse cell, a human cell, or a non-human primate cell.

95. 88. A non-human transgenic animal comprising the system of claim 2 and / or the transgenic cell of claim 87.

96. 96. The non-human transgenic animal of claim 95, which is a mouse or a non-human primate.

97. 100. A kit comprising the system of claim 2, the artificial expression construct of claim 38, the artificial expression construct of claim 60, the transgenic cell of claim 87, and / or the non-human transgenic animal of claim 95.

98. 61. A method of rescuing voltage-gated sodium channel function in a targeted cell population, comprising co-administering a therapeutically effective amount of the artificial expression construct of claim 38 and a therapeutically effective amount of the artificial expression construct of claim 60 to a sample or subject comprising the targeted cell population, thereby rescuing voltage-gated sodium channel function in the targeted cell population.

99. 99. The method of claim 98, wherein said co-administering comprises pipetting.

100. 100. The method of claim 99, wherein the pipetting is performed on a brain slice.

101. 101. The method of claim 100, wherein the brain slice comprises neurons.

102. 101. The method of claim 100, wherein the brain slice comprises GABAergic neurons or glutamatergic neurons.

103. 103. The method of claim 102, wherein the GABAergic neurons comprise whole GABAergic neurons, GABAergic neurons of the forebrain, GABAergic neurons of the hippocampus, or GABAergic neurons of the cortex.

104. 103. The method of claim 102, wherein the glutamatergic neurons comprise glutamatergic neurons of the forebrain.

105. 101. The method of claim 100, wherein the brain slice comprises a cell line.

106. 101. The method of claim 100, wherein the brain slice is a mouse, human or non-human primate brain slice.

107. 99. The method of claim 98, wherein said co-administration comprises administration to a living subject.

108. 108. The method of claim 107, wherein the living subject is a human, a non-human primate, or a mouse.

109. 108. The method of claim 107, wherein the living subject has been diagnosed with an SCN1A-associated seizure disorder and is therefore in need of rescue of voltage-gated sodium channel function.

110. The method of claim 109, wherein the SCN1A-associated seizure disorder comprises Dravet syndrome, myoclonic seizures, myoclonic-astatic epilepsy (MAE), refractory childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epileptic febrile seizures plus (GEFS+), infantile focal migrating partial seizures, Lennox-Gastaut syndrome, or West syndrome.

111. 108. The method of claim 107, wherein the living subject is a pediatric patient.

112. 108. The method of claim 107, wherein the living subject is under 4 years of age.

113. 108. The method of claim 107, wherein the living subject is a non-human transgenic animal.

114. 99. The method of claim 98, wherein the co-administration comprises intravenous injection, intraparenchymal injection into brain tissue, intracerebroventricular (ICV) injection, intracisternal (ICM) injection, or intrathecal injection.

115. An artificial expression construct comprising CN3252, CN3254, CN3683, CN3684, CN3251, CN3253, CN3677, CN3678, CN4541, CN4542, CN4217, CN4218, CN4642, or CN4643.

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