Rescue of voltage-gated sodium channels in inhibitory neurons

Abstract

To provide expression constructs, vectors, and methods that result in protein expression selectively within inhibitory neurons for the purpose of rescuing defective Nav1.1 channel function.SOLUTION: Provided is a method of treating Dravet Syndrome in a subject in need thereof, comprising intravenously administering a therapeutically effective amount of at least two adeno-associated viral (AAV) vectors, each viral vector comprising an expression construct comprising (i) an enhancer; (ii) a promoter; and (iii) a coding sequence that results in expression of a fragment of human SCN1A, the AAV vectors are associated with a capsid that crosses the blood brain barrier, and the human SCN1A fragments encoded by the coding sequences being expressed selectively within inhibitory GABAergic interneurons and result in rescue of Nav1.1 channel function within inhibitory GABAergic interneurons in need thereof following the intravenous administering.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 62 / 655,043 (filed April 9, 2018), 62 / 742,835 (filed October 8, 2018), and 62 / 810,281 (filed February 25, 2019), each of which is incorporated by reference in its entirety as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant number RF1MH114126 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field of Disclosure The present disclosure describes the selective rescue of voltage-gated sodium channel function in inhibitory neurons in need thereof. The rescued voltage-gated sodium channel function in inhibitory neurons can be used to treat disorders such as epilepsy, and more particularly, Dravet syndrome. [Background technology]

[0004] There are many neurological disorders that require urgent treatment. One class of such disorders is caused by the dysfunction of Nav1.1 sodium channels in inhibitory neurons. For example, epilepsy, infantile spasms, migraine, and autism spectrum disorder are associated with the dysfunction of Nav1.1 sodium channels.

[0005] Epilepsy is a neurological disorder that occurs when the brain exhibits a persistent tendency to have two or more epileptic seizures. An epileptic seizure is a temporary disruption of brain function due to abnormal, excessive, or simultaneous neural activity. Its manifestations include abnormal behavior, abnormal sensations, and sometimes periods of loss of consciousness.

[0006] Dravet syndrome (DS) is a rare and devastating form of intractable epilepsy that begins specifically in infancy. Initially, patients experience persistent seizures. During the second year, additional types of seizures begin to occur, accompanied by a decline in development. This results in poor development of language and motor skills.

[0007] Children with DS are prone to multiple seizures per day. Epileptic seizures result in a much higher mortality rate in individuals with DS. 10-16% of individuals diagnosed with DS die between the ages of 2 and 4. Additionally, patients are at risk for many associated conditions, including orthopedic and developmental problems, poor growth, sleep and circadian rhythm disorders, and chronic infections.

[0008] Of particular note, children with DS are particularly susceptible to episodes of status epilepticus. Status epilepticus is a condition characterized by a seizure lasting more than five minutes or multiple seizures occurring within five minutes of each other without regaining consciousness between seizures. This severe condition is classified as a medical emergency requiring immediate medical intervention, often including hospitalization. Sustained convulsive status epilepticus lasting more than 30 minutes can be fatal and can result in severe brain damage. Frequent hospitalizations of children with DS are clearly distressing not only to the patient but also to their families and caregivers. Because children with DS require constant management and frequent institutionalization, the cost of care for DS is also high.

[0009] Currently, many anticonvulsant treatments are available to reduce the incidence of seizures in DS patients, but the results obtained with such treatments are generally poor, and these treatments only result in partial cessation of seizures in many patients. Many of these anticonvulsant drugs, such as clobazam and clonazepam, have undesirable side effects, which are particularly acute in pediatric patients. Furthermore, some anticonvulsant drugs (especially sodium channel blockers) can worsen seizures.

[0010] Cell-type or cell-class-specific gene delivery using nonpathogenic viral delivery has shown increasing support for the treatment of a wide range of diseases. The inclusion of specific gene regulatory elements, such as specific promoters or enhancers, in the delivered vector has been beneficial for providing specificity to gene expression in specific target cell types. For example, Dimidschstein and colleagues (Nat Neurosci 19(12):1743-1749, 2016) developed an adeno-associated virus (AAV)-based viral delivery construct that resulted in the selective expression of a gene in the telencephalon, a cell type important in the treatment of epilepsy. This construct contained a 529-base pair (bp) enhancer sequence (termed mI56i or mDlx).

[0011] One significant drawback of using AAV as a selective gene delivery system is its severely restricted packaging capacity, which is particularly limiting for incorporating long gene regulatory elements. Furthermore, existing interneuron-specific AAV expression constructs result in weak expression in certain applications and are much less tolerant of transgene (e.g., therapeutic gene) expression than other, more commonly used proteins. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Dimidschstein and colleagues, Nat Neurosci 19(12):1743-1749,2016 Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, there remains a need in the art for shorter enhancer sequences that can confer rapid and strong expression of functional proteins in a cell type of choice. [Means for solving the problem]

[0014] The present disclosure provides expression constructs that result in unexpectedly rapid and high-level protein expression selectively in inhibitory neurons to rescue defects in Nav1.1 channel function. In certain embodiments, the present disclosure provides a concatemerized core of a human I56i enhancer and a gene encoding a voltage-gated sodium channel protein or a nucleotide sequence capable of rescuing defective Nav1.1 sodium channel function. The expression constructs can be used to reverse or improve dysfunctional Nav1.1 voltage-gated sodium channels in inhibitory neurons. In certain embodiments, the present disclosure provides treatment for sodium channel disorders by selectively delivering genes that allow voltage-gated sodium channels to function to inhibitory neurons. Administration of viral vectors containing these enhancers and genes results in the selective expression of proteins or nucleotide sequences that rescue Nav1.1 sodium channel function in inhibitory neurons. In certain embodiments, the therapeutic voltage-gated sodium channel genes in these viral vectors can be small and of bacterial origin, fitting within a single AAV construct, or larger and of human origin, allowing for the use of dual AAV construct delivery techniques, among other strategies. Treatable Nav1.1 sodium channelopathies include epilepsy, and more specifically, Dravet syndrome (DS). For example, as disclosed herein, administration of an expression construct results in therapeutic efficacy in treating DS in a well-established in vivo mouse model.

[0015] Many of the drawings presented herein are better understood in color, and applicants reserve the right to consider color versions of the drawings as part of the original application and to show color images of the drawings in later prosecutions. [Brief explanation of the drawings]

[0016] [Figure 1A] Figures 1A-1D. A pan-GABAergic viral enhancer labels fast-spiking interneurons in human organotypic slice cultures. (Figure 1A) Green fluorescence identifies cells expressing the CN1180 / DJ viral vector at 7 DIV / DPI (DIV: days in vitro, DPI: days post-infection). CN1180 contains a SYFP2 expression cassette under the control of the human DLX (hDLX) I12b enhancer and a minimal beta-globin promoter. Neurons were patch-clamped and later visualized using Alexa 594 backfill in the red channel. [Figure 1B] Figures 1A-1D. Pan-GABAergic viral enhancers label fast-spiking interneurons in human organotypic slice cultures. (Figure 1B) Morphology of patched cells from a single human case, sorted by the presence of YFP fluorescence, suggests that SYFP2-expressing cells are primarily interneurons. [Figure 1C] Figures 1A-1D. Pan-GABAergic viral enhancers label fast-spiking interneurons in human organotypic slice cultures. (Figure 1C) Electrophysiological parameters of YSP+ neurons suggest that these cells are primarily fast-spiking interneurons. [Figure 1D] Figures 1A-1D. A pan-GABAergic viral enhancer labels fast-spiking interneurons in human organotypic slice cultures. (Figure 1D) Post-hoc immunohistochemistry demonstrated that many YSP+ cells (arrows) express parvalbumin (PVALB), suggesting that these cells possess molecular characteristics of fast-spiking interneurons. [Figure 2] Figure 2 shows an exemplary vector for targeted expression of proteins in inhibitory neurons. CN1367-rAAV2-hI56i-minBglobin-His-NavMs-P2A-SYFP2-WPRE3-BGHpA. Symbols: hI56i—full-length human DLX I56i enhancer (SEQ ID NO: 1); minBglobin—minimal beta globulin promoter; His-NavMs—hexahistidine-tagged voltage-gated sodium channel from Magnetococcus marinus; P2A—self-cleaving peptide; SYFP2—Super Yellow Fluorescent Protein 2; WPRE3—Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element 3 (SEQ ID NO: 19); BGHpA—bovine growth hormone polyA sequence. [Figure 3] Viral CN1244 / PHP.eB.1011 genome copies were delivered intravenously (IV). PHP.eB encodes an AAV9-directed capsid, which allows efficient AAV translocation across the blood-brain barrier and allows AAV vector delivery in a brain-wide manner. This capsid differs from AAV by changing the amino acid sequence starting at residue 586: SAQA to SDGTLAVPFKA. The Gad2-T2A-nls-mCherry reporter expressed nearly all inhibitory neurons in the mouse brain (here, V1 visual cortex), and the delivered CN1244 / PHP.eB virus drove specific SYFP2 reporter activity in forebrain inhibitory neurons. [Figure 4A] Figures 4A and 4B. Comparison of CN1244 vs. CN1389 vs. CN1390. (Figure 4A) Schematic diagram of the three constructs: CN1244, CN1389, and CN1390 (CN1203 scAAV). Key: hI56i—full-length human DlxI56i enhancer (SEQ ID NO: 1); hI56icore—human DlxI56i enhancer core (SEQ ID NO: 2); minBG—minimal beta-globin promoter; SYFP2—Super Yellow Fluorescent Protein 2; WPRE3—Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element 3; BGHpA—bovine growth hormone poly(A) sequence; L-ITR and R-ITR—adeno-associated virus-2 (AAV2) inverted terminal repeats (ITRs). [Figure 4B] Figures 4A, 4B. Comparison of CN1244 vs. CN1389 vs. CN1390. (Figure 4B) Fluorography image showing the relative expression of SYFP2 from AAV vector constructs CN1244, CN1389, and CN1390. [Figure 5] In the cortex and hippocampus of young mice in slice cultures, CN1390 retains cell-type specificity of reporter expression for the pan-GABAergic neuronal population (marked with red fluorescence in these Ai75 het / Gad2-IRES-Cre mice) at 10 DIV / 10 DPI. [Figure 6A] Figures 6A-6E. CN1390 demonstrates rapid onset of transgene expression in human ex vivo brain slices. (Figure 6A) Time course of viral-mediated YFP expression after transduction of human brain slices with CN1390 / PHP.eB (left panel: 1 DIV / 1 DPI; middle panel: 3 DIV / 3 DPI; right panel: 6 DIV / 6 DPI). [Figure 6B] Figures 6A-6E. CN1390 demonstrates rapid onset of transgene expression in human ex vivo brain slices. (Figure 6B) Enlargement of the boxed area in Figure 6A, right panel. [Figure 6C] Figures 6A-6E. CN1390 demonstrates rapid onset of transgene expression in human ex vivo brain slices. (Figure 6C) High magnification image of virally labeled interneurons showing polarized morphology. [Figure 6D] Figures 6A-6E. CN1390 demonstrates rapid onset of transgene expression in human ex vivo brain slices. (Figure 6D) Examples of whole-cell recordings from YFP+ human interneurons labeled with four different viruses, showing different firing patterns in response to suprathreshold current injections. [Figure 6E] Figures 6A-6E. CN1390 demonstrates rapid onset of transgene expression in human ex vivo brain slices. (Figure 6E) Functional analysis of human neocortical interneuron firing patterns and electrical properties by patch clamp recording is feasible as early as 40 hours after CN1390 / PHP.eB virus infection. [Figure 7A]Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7A) Vectors for delivering bacterially derived epitope-tagged Nav genes (NavBacs). The Nav genes shown here are NavMs (from Magnetococcus marinus), NavBp (from Bacillus pseudofirmus), and NavSheP-D60N (from Shewanella putrifaciens with a genetically engineered D60N mutation). All of these examples contain an N-terminal epitope tag (hexahistidine in the case of CN1367, or 3xHA for CN1498, CN1499, and CN1500). hI56i refers to the full-length I56i enhancer of SEQ ID NO: 1; 3xhI56iCore refers to the concatemerized core of the enhancer (SEQ ID NO: 3). [Figure 7B] Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7B) Graded expression levels from NavBac vectors. [Figure 7C] Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7C) Weak but detectable expression from vector CN1367 in some Pvalb interneurons. [Figure 7D] Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7D) Trend towards seizure protection by vector CN1367. [Figure 7E] Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7E) Vector 1500 drives high-level expression in Pvalb+ and Pvalb- interneurons throughout the cortex. [Figure 7F]Figures 7A-7F. AAV vector reagents for reversing DS symptoms in Scn1a+ / - mice. (7F) Abundant production of HA-tagged NavBac in cell bodies and proximal processes is conferred by vectors 1498 and 1500, but not 1499. [Figure 8-1] Figure 8. Four example strategies for encoding and delivering the full-length human SCN1A protein despite exceeding the AAV packaging limit. The human SCN1A gene (hSCN1A) has a 6030-bp open reading frame, exceeding the 4.7-kb packaging limit in AAV vectors. One approach to overcoming this size limit is to split it into two for delivery. Strategies 1 and 2 utilize homology-driven recombination between two separate AAV vector genomes (regions of homology are indicated by dashed lines) to enable complete open reading frame reconstitution in cotransfected cells. In contrast, strategies 3 and 4 add trans-splicing elements (synthetic introns indicated by dots), potentially increasing open reading frame reconstitution and thereby full-length protein expression. Strategies 1 and 3 utilize SYFP-P2A- and 3xHA-tagged hSCN1A proteins, allowing for easy detection of both portions of the protein by fluorescence and immunohistochemistry. On the other hand, strategies 2 and 4 utilize untagged hSCN1A protein and a vertebrate promoter to ensure reduced immunogenicity of the delivered transgene and encoded protein. Another strategy utilizes multiple vectors, each encoding a different segment or subunit of the SCN1A protein. The protein subunits may then self-assemble after expression, either naturally or via engineered cysteine ​​or other binding domains. [Figure 8-2]Figure 8. Four example strategies for encoding and delivering the full-length human SCN1A protein despite exceeding the AAV packaging limit. The human SCN1A gene (hSCN1A) has a 6030-bp open reading frame, exceeding the 4.7-kb packaging limit in AAV vectors. One approach to overcoming this size limit is to split it into two for delivery. Strategies 1 and 2 utilize homology-driven recombination between two separate AAV vector genomes (regions of homology are indicated by dashed lines) to enable complete open reading frame reconstitution in cotransfected cells. In contrast, strategies 3 and 4 add trans-splicing elements (synthetic introns indicated by dots), potentially increasing open reading frame reconstitution and thereby full-length protein expression. Strategies 1 and 3 utilize SYFP-P2A- and 3xHA-tagged hSCN1A proteins, allowing for easy detection of both portions of the protein by fluorescence and immunohistochemistry. On the other hand, strategies 2 and 4 utilize untagged hSCN1A protein and a vertebrate promoter to ensure reduced immunogenicity of the delivered transgene and encoded protein. Another strategy utilizes multiple vectors, each encoding a different segment or subunit of the SCN1A protein. The protein subunits may then self-assemble after expression, either naturally or via engineered cysteine ​​or other binding domains. [Figure 9]Full-length human SCN1A expression driven by a two-part AAV viral vector system (upper panel): A model of the two-part vector system. The upper vector (1504) contains concatemerized 3xhI56iCore (SEQ ID NO: 3) and a promoter (minCMV) element to drive expression in all inhibitory neurons (including both Pvalb+ and Pvalb- inhibitory neurons). The driven transgene contains SYFP2 linked by a P2A tag to the N-terminal region of human SCN1A, which contains 604 bp of homology to the C-terminal region shown in the lower vector 1512. The lower vector 1512 also contains a C-terminal 3xHA tag and 3'UTR regulatory sequences (WPRE3 and polyA site). (lower panel): Both AAV viral vectors were packaged into viral particles with PHP.eB capsids, and both were delivered intravenously to C57Bl / 6 mice. After 21 days, mouse brains were harvested, fixed, and processed for immunofluorescence with anti-GFP (targeting SYFP2), anti-HA, and anti-Pvalb antibodies to detect overlapping of transgene-expressing cells with Pvalb+ inhibitory neurons (cells particularly important in DS symptomology). Many inhibitory neurons, including both Pvalb+ and Pvalb- inhibitory neurons, were highly labeled with the N-terminal SYFP2+ tag. Some of these SYFP2+ cells also expressed HA (the fully intact SCN1A protein product expressed by these GFP+HA+ cells) (arrows). Vertical arrows indicate Pvalb+ interneurons expressing human SCN1A, and horizontal arrows indicate Pvalb- interneurons expressing human SCN1A. These SCN1A-expressing cells are found throughout the forebrain (both the neocortex and hippocampus [herein referred to as the gyrus]), which includes brain regions known to be important in many epilepsies and known to be dysfunctional in DS. [Figure 10]Figure 10. The CN1500 rAAV vector substantially ameliorates febrile seizures in Scn1a+ / − mice. The febrile seizure assay is shown as the internal temperature at which seizures are first detected (top). Circles represent Scn1a+ / − mice not transduced with AAV, and diamonds represent animals transduced with CN1500. Large dots and error bars represent the mean + / - SEM for each animal group (bottom). The trend of the same data to remain seizure-free at different temperatures is shown using Kaplan-Meier curves as the percentage of mice in each group. [Figure 11-1] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-2] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-3] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-4] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-5] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-6] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-7] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-8] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-9] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-10] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-11] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-12] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-13] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-14] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-15] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-16]Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-17] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-18] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-19] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-20] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-21] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-22] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-23] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-24] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-25] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-26] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-27] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-28] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-29] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-30] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-31] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-32] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-33] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-34] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-35] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-36] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-37]Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-38] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-39] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-40] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-41] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-42] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-43] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-44] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-45] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-46] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-47] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-48] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-49] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-50] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-51] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-52] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-53] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-54] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-55] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-56] Figure 11. Example sequences 1-57 supporting the present disclosure. [Figure 11-57] Figure 11. Example sequences 1-57 supporting the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description In vertebrates, voltage-gated sodium channels (Nav) are heteromeric protein complexes composed of a large central pore composed of alpha subunits and smaller auxiliary beta subunits that regulate the dynamics and intracellular distribution of the pore. The alpha subunits are encoded by a family of nine distinct genes (SCN1A-SCN11A), and the beta subunits are encoded by four distinct genes (SCN1B-SCN4B), each of which exhibits cell- and tissue-specific expression patterns. Each of the nine distinct alpha subunit genes disseminates a distinct Nav channel complex, creating nine distinct subtypes (Nav1.1-Nav1.9). These nine Nav channels exhibit tissue-specific localization and functional differences (see Goldin, (2001) Annu Rev Physiol 63:871-94; and Yu et al., (2003) J. Neurosci 23:7577-758).

[0018] Navs are central to neuronal function and are responsible for initiating the rapid increase in potency in excitable nervous system cells, and Nav1.1 drives this activity in many cells. Nav1.1 channels contain the SCN1A alpha subunit and are expressed in nearly all neurons, with high levels in GABAergic neurons. Nav1.2 channels contain the SCN2A subunit, Nav1.3 channels contain the SCN3A subunit, Nav1.4 channels contain the SCN4A subunit, Nav1.5 channels contain the SCN5A subunit, Nav1.6 channels contain the SCN8A subunit, Nav1.7 channels contain the SCN9A subunit, Nav1.8 channels contain the SCN10A subunit, and Nav1.9 channels contain the SCN11A subunit.

[0019] There are many neurological disorders for which treatment is urgently needed. One class of such disorders results from dysfunction of Nav1.1 sodium channels in inhibitory neurons. For example, the following disorders and conditions have been associated with malfunction of the Nav1.1 sodium channel: epilepsy (Dravet syndrome (DS), generalized epilepsy with febrile seizures plus (GEFS+), borderline DS, intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), focal and generalized epilepsy of unknown etiology, myoclonic astatic epilepsy (Doose syndrome), Lennox-Gastaut syndrome, and severe multifocal infantile epilepsy (Gambardella A et al. Epilepsia. 2009, 50 Suppl 5:20-3)), West syndrome, also known as infantile spasms (Harkin LA et al. Brain, 2007, 130(3)843-852), rare cases of common migraines, e.g., familial hemiplegic migraine 3 (FHM3), Panayiotopoulos syndrome (Livingston et al. JHet al.J Child Neurol.2009,24(4):503-8), familial autism (Weiss LA et al.2003,Molecular Psychiatry,8(2),186-194), sporadic autism spectrum disorder (ASD) (Nat Genet.2011 43(6):585-9), Rasmussen's encephalitis, also known as chronic focal encephalitis, or CFE (Ohmori I.et al.,Epilepsy.2008 49(3):521-6), Alzheimer's disease (Scharfman HEEpilepsy Curr.2012 12(5):178-183), and cerebral ischemia-reperfusion (Yao C.et al.,Neurotox Res.2002;4(1):67).

[0020] The underlying cause of epilepsy is thought to arise from a deficit in the excitation-inhibition (E / I) balance in cortical circuits. Forebrain GABAergic interneurons are the first source of inhibition in the telencephalon, and a variety of lines of evidence support their importance in epilepsy. DS, specifically, is a severe form of childhood epilepsy primarily caused by SCN1A haploinsufficiency. It is characterized by severe and frequent seizures (sometimes up to 100 per day), developmental delays, and a mortality rate of up to 16%. DS affects nearly 1 in 20,000 births in both the United States and Europe, with an estimated 10,000+ individuals affected on both continents (Wu et al., Pediatrics 136, e1310-e1315, 2015). Heterozygous loss-of-function mutations in SCN1A, the gene encoding the pore forming subunit of the voltage-gated sodium channel Nav1.1, are the primary cause of DS (Catterall et al., J. Physiol. 588, 1849-1859, 2010; Claes et al., Hum. Mutat. 21, 615-621, 2003; Fujiwara, Epilepsy Res. 70 Suppl 1, S223-230, 2006; Verbeek et al., Epilepsy Behav. EB 47, 39-44, 2015). Genetic models of DS have established the pathophysiology of the disease (Han et al., Nature 489, 385-390, 2012; Kalume et al., J. Clin. Invest. 123, 1798-1808, 2013; Kalume et al., J. Neurosci. 27, 11065-11074, 2007; Kalume et al., Neurobiol. Dis. 77, 141-154, 2015; Oakley et al., Proc. Natl. Acad. Sci. 106, 3994-3999, 2009; Cheah et al., Proc. Natl. Acad. Sci. 109, 14646-14651, 2012; Han et al. al., Proc. Natl. Acad. Sci. 109, E368-E377, 2012).Mouse models of DS have reduced Nav1.1 function due to heterozygous deficiency of Scn1a and exhibit key phenotypes of DS, including febrile seizures, anxiety, and sleep debt (Han et al., Nature 489, 385-390, 2012; Kalume et al., J. Clin. Invest. 123, 1798-1808, 2013; Kalume et al., Neurobiol. Dis. 77, 141-154, 2015; Oakley et al., Proc. Natl. Acad. Sci. 106, 3994-3999, 2009; Tai et al., Proc. Natl. Acad. Sci. 111, E3139-E3148, 2014; Yu et al., Nat. Neurosci. 9, 1142-1149, 2006).

[0021] DS is a disease of forebrain interneurons. Global Scn1a deletion reduces sodium currents and the exitability of GABAergic interneurons without detectable effects on excitatory neurons (Catterall et al., J. Physiol. 588, 1849-1859, 2010; Kalume et al., J. Neurosci. 27, 11065-11074, 2007; Cheah et al., Proc. Natl. Acad. Sci. 109, 14646-14651, 2012; Tai et al., Proc. Natl. Acad. Sci. 111, E3139-E3148, 2014; Yu et al., Nat. Neurosci. 9, 1142-1149, 2006; Ogiwara et al. (e.g., J. Neurosci. 27, 5903-5914, 2007; Rubinstein et al., Brain 138, 2219-2233, 2015; Mistry et al., Neurobiol. Dis. 65, 1-11, 2014). Conditional deletion of Scn1a in the forebrain or interneurons using a specific Cre driver qualitatively reproduces the core symptoms of DS, whereas excitatory neuron-specific deletion does not result in a detectable phenotype (Cheah et al., Proc. Natl. Acad. Sci. 109, 14646-14651, 2012). In addition, Pvalb + or Sst + Targeted deletion of Scn1a in interneuron classes demonstrates that dysfunction in each class (separately or in combination) contributes distinctly to the pleiotropic phenotype of DS, as does Pvalb. +These findings demonstrate that DS cells exert a greater effect than GABAergic inhibition (Rubinstein et al., Brain 138, 2219-2233, 2015; Dutton et al., Neurobiol. Dis. 49, 211-220, 2013). Consistent with these mouse models, patients with DS exhibit reduced GABAergic inhibition and no changes in glutamatergic neuronal excitability after transcranial stimulation (Stern et al., Neurology 88, 1659-1665, 2017). Thus, both mouse and human studies strongly suggest that DS is a disease of forebrain interneurons caused by pathogenic loss-of-function mutations in SCN1A. Based on this insight, we hypothesize that targeted introduction of functional voltage-gated sodium channels with properties similar to Nav1.1 in forebrain GABAergic interneurons may ameliorate or eliminate DS symptoms through rescue of their physiological function. For an exemplary method for electrophysiologically and phenotypically characterizing DS in a mouse model, see Figure 1 of Rubinstein et al., Brain 138(Pt 8):2219-33, 2015.

[0022] Cell-type- or class-specific gene delivery using nonpathogenic recombinant adeno-associated viruses (rAAVs) has shown increasing promise as a therapeutic approach for a wide range of diseases. The inclusion of one or more cis-acting DNA regulatory elements (e.g., specific promoters or enhancers) in rAAVs is beneficial for providing specificity for expression within specific target cells, including cell types or cell classes within the brain. For example, Dimidschstein and colleagues (Nat Neurosci 19(12):1743-1749, 2016) developed an rAAV that directed selective gene expression in GABAergic interneurons within the telencephalon. The rAAV contains a 529 base pair (bp) enhancer sequence (termed mI56i or mDlx) from the distal-less homeobox 5 and 6 (Dlx5 / 6) gene, which is naturally expressed by forebrain GABAergic interneurons during embryonic development (Zerucha et al., J. Neurosci. 20(2):709-721, 2000). The construct developed by Dimidschstein et al. is available on Addgene as ID#83900 (in this construct, the enhancer drives eGFP expression). Constructs utilizing additional mouse or human I56i enhancers to drive various transgenes are also available through Addgene, e.g., plasmid ID#83899 (driving GCaMP6f expression), 83898 (driving ChR2-mCherry expression), 83895 (driving Cre recombinase-dependent eGFP expression), 89897 (driving bicistronic hM3Dq and nls-dTomato expression), 83896 (driving bicistronic hM4Di and nls-dTomato expression), and 83894 (driving cre recombinase-dependent tdTomato expression). See also U.S. Patent Application Publication US2018 / 0078658.Furthermore, the mDlx enhancer has previously been used to reliably target reporter genes in a pattern closely resembling the normal pattern of Dlx5 / 6 expression during embryonic development (Zerucha et al., J Neuroscience 20:709-721, 2000; Stuhmer et al., Cerebral Cortex 12:75-85, 2002; Stenman et al., J Neuroscience 23:167-174, 2003; Monory et al., Neuron. 51:455-455, 2006; Miyoshi et al., J Neuroscience 30:1532-1594, 2010).

[0023] One significant drawback of using rAAV as a selective gene delivery system is its severely restricted packaging limitations, which specifically limit the insertion of long gene regulatory elements. Furthermore, existing interneuron-specific rAAV expression constructs provide weak expression in certain applications and are less tolerant than GFP for transgene expression (e.g., therapeutic genes). Therefore, there remains a need in the art for even shorter enhancer sequences that can provide cell-specific expression of Nav1.1 restoration-of-function proteins (specifically, expression in neurons, such as inhibitory interneurons). There is also a need for genetic elements that provide stronger expression and / or accommodate a broader selection of reporter or other target genes in a wider variety of expression environments.

[0024] The present disclosure provides expression constructs that result in high-level protein expression in inhibitory neurons for the purpose of rescuing voltage-gated sodium channel function, which is selective to the targeted inhibitory neurons and provides efficacy for treating DS in well-established in vivo mouse models of the disease (described in detail in Kalume et al., J. Clin. Invest. 123, 1798-1808, 2013; and Oakley et al., Proc. Natl. Acad. Sci. 106, 3994-3999, 2009). In this model, mice may be implanted with electroencephalography (EEG) and electromyography (EMG) electrodes using approaches described in Kalume et al., J. Clin. Invest. 123, 1798-1808, 2013; Kalume et al., Neurobiol. Dis. 77, 141-154, 2015; and Oakley et al., Proc. Natl. Acad. Sci. 106, 3994-3999, 2009. After recovery, animals may be continuously monitored by video, EEG, and EMG. During the testing procedure, the mouse's temperature may be increased in 0.5°C increments until generalized tonic-clonic seizures occur or the core temperature reaches 42°C. Kaplan-Meier curves for temperature-induced seizure susceptibility may be generated for each treatment group, and the mean body temperature at seizure onset may be computed. Chi-squared tests can be fitted to Kaplan-Meier plots of seizure susceptibility, and unpaired two-tailed tests confirm differences between mean temperatures of seizure occurrence.

[0025] In certain embodiments, the expression construct comprises a non-naturally occurring enhancer element sequence, which, when used to drive a heterologous coding sequence to drive Nav1.1-associated sodium channelopathy, exhibits strong and rapid interneuron-specific expression. In certain embodiments, the non-naturally occurring enhancer element comprises multiple copies of a shortened core portion of the human or mouse I56i enhancer (SEQ ID NO: 3). Indeed, the present disclosure may include concatenated cores of I56i enhancers from any species, as long as the concatenated cores result in selective expression of a functional protein in inhibitory neurons. For example, the zebrafish I56i enhancer is presented as SEQ ID NO: 5, and the core of the zebrafish I56i enhancer is presented as SEQ ID NO: 6. In a detailed embodiment, the non-naturally occurring enhancer element comprises a concatemer of three copies of the human, mouse, or zebrafish I56i core (e.g., as shown in SEQ ID NO: 3 and column number 7). Further embodiments include, for example, tandemly arranged 2x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x copies of the I56i core sequence of SEQ ID NO: 2 or SEQ ID NO: 6. Particular embodiments can include concatemerized hybrids of SEQ ID NO: 2 and SEQ ID NO: 6 (e.g., 2-6-2, 6-2-6, 2-2-6, 6-6-2).

[0026] In certain embodiments, the concatemerized core of the I56i enhancer is used to minimize size and enhance expression of proteins or nucleotide sequences that rescue voltage-gated sodium channel function. The synthetic 3x human DLXI56i core enhancer (also known as 3xhI56iCore; SEQ ID NO: 3) is shorter than the original full-length enhancer sequence reported in Dimidschstein et al. (NatNeurosci 19(12):1743-1749, 2016), even though the 3x concatemer is a highly conserved core of the enhancer. When used to construct a heterologous expression cassette to drive expression of a protein that rescues Nav1.1 sodium channel function, this concatemerized core enhancer provides more room for enhancer-bound cargo genes, which is highly desirable in gene therapy vectors.

[0027] The compact size of the enhancer core and the multiple copies concatenated together resulted in unexpectedly strong peak transgene expression in forebrain interneurons after viral transduction of mouse and human brain tissue (see Figures 4B and 5). The onset was also surprisingly rapid (see Figures 6A and 6B), resulting in faster and higher expression compared directly with viruses packaged with the enhancer in, for example, Addgene plasmid #83900. The increase in expression was linear and not simply a tripling of the levels driven by the full-length enhancer (SEQ ID NO: 1). Thus, the concatenated I56i core enhancer also represents a novel and improved viral (and other) vector, such as the vector described herein, that may demonstrate better performance in treating Nav1.1 sodium channelopathy. These are particularly useful for achieving transgene expression in inhibitory brain cell types, such as neocortical GABAergic interneurons, in a wide range of animal species.

[0028] Thus, the present disclosure provides expression constructs, vectors, and methods useful for restoring or alleviating dysfunction of the voltage-gated sodium channel Nav1.1. In certain embodiments, the present disclosure provides for the treatment of Nav1.1 channelopathy by selectively delivering a gene that provides a protein or nucleotide sequence that allows for the rescue of voltage-gated sodium channel function to inhibitory interneurons with impaired Nav1.1 activity.

[0029] In certain embodiments, genes encoding proteins that rescue voltage-gated sodium channel function when Nav1.1 is impaired are bacterial Nav genes (NavBacs) (Nguyen et al., Nat. Commun. 7, 13132, 2016; Sula et al., Nat. Commun. 8, 14205, 2017; DeCaen et al., eLife 3, e04387, 2014). Examples of specific NavBacs are NavSheP, NavBp, and NavMs, which are three Nav proteins from three distinct bacterial species. For representative experimental protocols and data demonstrating that bacterial voltage-gated sodium channels can confer sodium conductivity to non-excitable cells, see Nguyen et al., Nature Communications 18(7), 13132, 2016, specifically Supplementary Figure 8.

[0030] NavBac proteins can also be mutagenesis-enhanced or modified to enhance or alter activity as needed, as in NavSheP-D60N. Additionally, these proteins can be engineered to incorporate tags or other fusion proteins, such as those containing His-NavMs (including an N-terminal hexahistidine tag) or an N-terminal 3xHA tag, or other epitope tags, fluorescent protein tags, or any other protein tag, for detection as needed. In certain embodiments, the expression construct does not encode an immunogenic component. In certain embodiments, the expression construct does not include or encode an immunogenic component.

[0031] In certain embodiments, the gene encoding a protein that restores voltage-gated sodium channel function is a gene encoding SCNA1 (eg, human SCNA1 or mouse Scna1).

[0032] In certain embodiments, therapeutic treatments based on intravenous, retro-orbital, intraspinal and / or intrathecal administration of viral vectors result in selective expression in targeted inhibitory neurons.

[0033] In a specific embodiment, the AAV viral vector CN1500 is provided herein, which is a recombinant AAV expressing the transgene SYFP2-P2A-NavSheP-D60N, which upon translation cleaves into two proteins: SYFP2 (reporter) and NavSheP-D60N. As shown, NavSheP-D60N (Nguyen et al., Nat. Comm. 7:13132, 2016) is a bacterially derived, modified voltage-gated sodium channel engineered to improve channel kinetics and codon-optimized for enhanced expression in mammalian cells. Transgene expression levels are elevated by the addition of a WPRE3 element, and transcription is terminated by the bovine growth hormone polyadenylation sequence (BGHpA). Transgene expression, mediated by the 3xhI56iCore synthetic enhancer directly 5' of the cytomegalovirus (CMV) minimal promoter, is high and restricted to inhibitory cells in forebrain structures, including the cortex and hippocampus. Furthermore, the therapeutic transgene NavSheP-D60N may be tagged with an HA epitope tag to verify protein expression and precise protein localization. Therapeutic efficacy has been demonstrated in mouse models of DS. In certain embodiments, the HA epitope tag may be omitted from CN1500 and other vector designs described herein.

[0034] The human SCNA1 gene is relatively large and may benefit from administration from a vector that can accommodate a larger cargo than AAV, or by using a two-AAV vector administration strategy. A number of different strategies may be utilized to split the large open reading frame between two AAV vectors. Four major strategies for splitting the large hSCN1A open reading frame are shown in Figure 8, although several more can be envisioned.

[0035] In certain embodiments, a two-vector administration strategy may split the coding sequence between two vectors. In certain embodiments, the first vector may encode the N-terminal portion of the protein, including the promoter and enhancer. The second vector may encode the C-terminal portion of the protein and may include a termination signal and poly(A) signal. The portions of the protein encoded by each vector overlap, creating a region of homology. Example 2, Figure 8 Strategy 1, and Figure 9 show examples of such two-vector systems that achieve selective expression of human SCN1A protein in inhibitory neurons. The 604-base pair (bp) region of homology is indicated by a diagonal dashed line in Figure 9. However, as will be understood by those skilled in the art, a wide range of numbers of bp of homology may be selected. In certain embodiments, the bp region of homology ranges from less than 75 bp to less than 1500 bp. Additionally, various other components and configurations may be appropriate for two-vector systems, as described in more detail elsewhere herein in comparison to single-vector systems. Another possible configuration includes synthetic intron elements that provide regions of homology and splicing donor and acceptor sites, resulting in more efficient final protein rearrangement and higher levels of protein expression.

[0036] Furthermore, additional strategies for providing exogenous voltage-gated sodium channel activity to inhibitory neurons lacking such activity may also be used. For example, certain embodiments may utilize expression constructs encoding artificial transcription factors that increase expression of endogenous functional copies of SCN1A.

[0037] In certain embodiments, the expression construct may encode a molecule that increases the prevalence of functional SCN1A mRNA molecules, for example, through post-transcriptional positive regulation of splicing and / or stability.

[0038] In certain embodiments, a strategy may be used to recover the full-length hSCN1A transcript through trans-splicing of two separate hSCN1A transcripts.

[0039] In certain embodiments, the expression construct (e.g., AAV) can deliver the hSCN1A protein as several (two or four) subunit ORFs delivered by two or four separate vectors.

[0040] Aspects of the present disclosure are now illustrated by the following further selections and details: (i) expression constructs and vectors; (ii) compositions for administration; (iii) methods of use; (iv) kits and supplementary commercial packaging; (iv) exemplary embodiments; and (v) experimental examples.

[0041] (i) Expression Constructs & Vectors. The expression constructs disclosed herein comprise (i) a concatenated core of I56i enhancer sequences that direct the selective expression of coding sequences in inhibitory neurons, (ii) a coding sequence that is expressed to produce a protein or nucleotide sequence that rescues voltage-gated sodium channel function in cells in need of rescue, and (iii) a promoter. The expression construct may also include other regulatory elements if necessary or beneficial. In certain embodiments, the expression construct is an isolated polynucleotide.

[0042] In certain embodiments, an "enhancer" or "enhancer element" is a cis-acting sequence that increases the level of transcription associated with a promoter, can function in either orientation relative to the promoter and the coding sequence being transcribed, and can be located upstream or downstream from the promoter or the coding sequence being transcribed. Art-recognized methods and techniques exist for measuring the function of enhancer element sequences. By way of example, specific methods for determining or measuring the function of the I56i enhancer are described in Dimidschstein et al. (Nat Neurosci 19(12):1743-1749, 2016) and U.S. Patent Application Publication US2018 / 0078658. Specific examples of enhancer sequences include the human full-length I56i enhancer (SEQ ID NO: 1), hI56 core (SEQ ID NO: 2), and 3xhI56i core (SEQ ID NO: 3), as well as their mouse and zebrafish orthologs.

[0043] In certain embodiments, an inhibitory neuron-specific enhancer is an enhancer that is uniquely or preferentially used in inhibitory neurons. An inhibitory neuron-specific enhancer increases gene expression in inhibitory neurons without substantially affecting gene expression in other cell types, such as non-inhibitory neurons or glial cells, and thus has neuron-specific transcriptional activity. In some cases, there may be some low-level expression in other cell types, but such expression is substantially lower than in inhibitory neurons, e.g., less than 1%, or 1%, 2%, 3%, 5%, 10%, 15%, or 20% of the expression level in inhibitory neurons. In certain embodiments, interneurons are the only cell type that express the correct combination of transcription factors that bind to the concatenated core of the I56i enhancer to drive gene expression.

[0044] In certain embodiments, selective expression in inhibitory neurons is demonstrated by expression in greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 100% or more of non-inhibitory neurons. In certain embodiments, selective expression in inhibitory neurons is demonstrated by expression in inhibitory neurons and the absence of detectable expression in non-inhibitory neurons.

[0045] In certain embodiments, selective expression in GABAergic interneurons is demonstrated by greater than 10% expression, greater than 20% expression, greater than 30% expression, greater than 40% expression, greater than 50% expression, greater than 60% expression, greater than 70% expression, greater than 80% expression, greater than 90% expression, greater than 100% expression, or more than non-GABAergic interneurons. In certain embodiments, selective expression in GABAergic interneurons is demonstrated by expression in GABAergic interneurons and the absence of detectable expression in non-GABAergic interneurons.

[0046] In certain embodiments, a class of inhibitory neurons can be identified based on Pvalb expression, as described in connection with Figures 1, 7C, 7E, and 8. In certain embodiments, GABAergic interneurons can be distinguished from other cell types in the adult cortex by expression of the genes Gad2 and Gad1, and GABAergic interneurons can be distinguished from glutamatergic excitatory neurons by the presence of GABA. In the adult striatum, GABAergic interneurons can be distinguished from medium spiny neurons by expression of the gene Nkx2.1 (e.g., Rudy et al., Devel Neurobio 71, 45-61 (2011)); Kepecs. & Fishell, Nature 505, 318-326 (2014).

[0047] In certain embodiments, the coding sequence or nucleotide sequence encodes a protein that rescues voltage-gated sodium channel function. In certain embodiments, the coding sequence further encodes a reporter protein. When the coding sequence encodes a protein or nucleotide sequence that rescues voltage-gated sodium channel function and a reporter protein, it may further encode a skipping element, such as a self-cleaving peptide or an internal ribosome entry site (IRES) sequence.

[0048] Examples of proteins that restore voltage-gated sodium channel function include NavShep, NavShep-D60N, NavBp, NavMs, and hSCN1A, as well as their epitope-tagged variants 3xHA-NavShep-D60N, 3xHA-NavBp, 3xHA-NavMs, His-NavMs, and hSCN1A-3xHA.

[0049] Examples of coding sequences that encode proteins that rescue voltage-gated sodium channel function include SEQ ID NOs: 9-16.

[0050] Moreover, as indicated above, there are additional strategies that can be used to provide exogenous voltage-gated sodium channel activity to inhibitory neurons that are deficient in that activity. For example, in certain embodiments, SCN1A + / -To increase the expression of an endogenous functional copy of SCN1A (or other SCN_A family genes) in patients (e.g., Dravet patients) or other patients with deficiencies in other voltage-gated sodium channels, enhancer-vectors (e.g., AAV) may encode artificial transcription factors, which may contain specific DNA-binding domains linked to a general transcription activation domain. DNA-binding domains can be engineered from Cas9-associated genes using CRISPR activation technology (as described in Matharu et al., 2019, Science, 363(6424), 186-194, where the Cas protein is engineered to lack nuclease activity), or from other conventional DNA-sensing proteins such as TALE transcription factors (TFs) (Morbitzer et al., 2010, Proc. Nat. Acad. Sci., 107(50), 21617-21622) or zinc finger TFs (Gersbach et al., 2014, Acc. Chem. Res, 47(8), 2309-2318). Further information and options regarding CRISPR and other targeted gene binding (and optionally editing) systems and components are provided below.

[0051] In certain embodiments, enhancer-vectors can encode molecules that increase the prevalence of functional SCN1A mRNA molecules, for example, through positive regulation of post-transcriptional splicing and other stability, ultimately resulting in increased functional voltage-gated sodium channel activity. This can be achieved by AAV delivery of antisense RNA oligonucleotide molecules to increase splicing (Hsiao et al., 2016, EBioMedicine. 9, 257-277) or by preventing negative regulation of potential microRNAs.

[0052] In certain embodiments, strategies can be used to restore full-length hSCN1A transcripts through splicing of the two-part hSCN1A transcript, which can be accelerated through the use of strong synthetic splicing donors and splicing acceptors in a two-vector system and through the use of different ITRs (ITR2 and ITR5) that induce proper juxtaposition of the two distinct genomes in transduced cells (McClements ME, et al., 2017, Yale J. Biol. Med. 90(4):611-623).

[0053] In certain embodiments, enhancer-vectors (e.g., AAV) can deliver the hSCN1A protein as several (two or four) subunit ORFs delivered by separate vectors. Nav channels exhibit pseudo-fourfold internal symmetry due to four similar internal domains within the alpha subunit (Shen et al., Science 363(6433), 1303-1308), indicating that this protein is suitable for delivery in two or four parts. As previously indicated, subunit assembly may occur naturally and / or may be facilitated by the inclusion of engineered cysteines or other binding domains.

[0054] In certain embodiments, due to the pseudosymmetry of its internal four domains, hSCN1A can be delivered in four fragments encoded on four different vectors, which can self-assemble into a tetramer that is functionally and chemically equivalent to the functional NaV1.1 alpha subunit, except for the breakpoints in the surface loops. Alternatively, one fragment can be delivered by one vector, which can self-assemble into a homotetrameric complex reminiscent of the NaV1.1 alpha subunit consisting entirely of human sequences. Either strategy can deliver human non-immunogenic biological molecules to inhibitory cells in order to rescue NaV1.1 channel function in neurons that lack voltage-gated sodium channel activity.

[0055] An additional strategy can be used to shorten the hSCN1A ORF to a smaller (eg, AAV-compatible) size (<4.7 kb).

[0056] Examples of reporter proteins include fluorescent proteins such as yellow fluorescent molecules such as SYFP2, Citrine, PhiYFP, and ZsYellow1; red fluorescent molecules such as mCherry, mRuby, Jred, and AsRed2; green fluorescent molecules such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), avGFP, ZsGreen, and mAzamiGreen; orange fluorescent molecules such as mOrange and mKusabira-Orange; blue fluorescent molecules such as Sapphire, mKalama1, EBFP2, and Azurite; cyan fluorescent molecules such as Cerulean and mTurquoise; and far-red proteins such as mPlum and mNeptune.

[0057] GFP, consisting of 238 amino acids (26.9 kDa), was first isolated from the jellyfish Aequorea victoria / Aequorea aequorea / Aequorea forskalea and emits green fluorescence when exposed to blue light. GFP 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 portion of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a major excitation peak at 498 nm. Due to its widespread use and growing demand from researchers, many different variants of GFP have been genetically engineered. The first significant improvement was a single point mutation (S65T) reported by Roger Tsien in Nature in 1995. This mutation dramatically improved the optical properties of GFP, resulting in increased fluorescence, photostability, and a shift in the main excitation peak to 488 nm while maintaining the emission peak at 509 nm. Addition of a 37°C folding efficiency (F64L) point mutation to this scaffold resulted in enhanced GFP (EGFP). EGFP has an extinction coefficient (denoted ε), also known by its optical cross section of 9.13E-21 m / mol, also quoted as 55,000 L / (mol·cm). Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature, even when fused to a misfolded peptide, was reported in 2006.

[0058] Yellow fluorescent protein (YFP) is a genetic mutation of green fluorescent protein derived from Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm.

[0059] An example of a self-cleaving peptide is the 2A peptide, which results in the production of two proteins from one mRNA. 2A sequences are short (e.g., 20 amino acids), making them more useful in size-constrained constructs. Specific examples include P2A, T2A, E2A, and F2A. In certain embodiments, the expression construct contains an internal ribosome entry site (IRES) sequence. The IRES directs ribosomes to begin translation at a second internal site on the mRNA molecule, resulting in the production of two proteins from one mRNA.

[0060] Coding sequences encoding the proteins described herein can be obtained from publicly available databases and publications. The coding sequences can further contain various sequence polymorphisms, mutations, and / or sequence variants, where such modifications do not affect the function of the encoded protein. The terms "encode" or "encoding" refer to the property of a nucleic acid sequence, such as a vector, plasmid, gene, cDNA, mRNA, etc., to serve as a template for the synthesis of other molecules, such as proteins.

[0061] The term "gene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term may further include all introns and other DNA sequences spliced ​​from the mRNA transcript, along with variants resulting from alternative splicing sites. A sequence may also include degenerate codons of the reference sequence(s), which may be introduced to provide codon preference in a particular organism or cell type.

[0062] Promoters may include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters may include strong promoters, weak promoters, constitutive promoters, and / or inducible promoters. Inducible promoters direct 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 affect transcription from the promoter. Specific examples of promoters include minB globin, CMV, minCMV, SV40 immediate early promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.

[0063] In certain embodiments, the expression construct is provided within a vector. The term vector refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally linked to, e.g., integrated into, a vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within a cell, or may contain a sequence that allows integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0064] Viral vectors are widely used and refer to nucleic acid molecules containing virally derived nucleic acid elements 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 containing structural and functional genetic elements or portions thereof derived primarily from AAV. The term "retroviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or portions thereof derived primarily from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or portions thereof derived primarily from lentiviruses, etc. The term "hybrid vector" refers to a vector containing structural and / or functional genetic elements derived from more than one type of virus.

[0065] Adenovirus. An "adenovirus vector" refers to a construct containing sufficient adenovirus sequences to (a) support packaging of an expression construct and (b) express a coding sequence cloned therein in either the sense or antisense orientation. Recombinant adenovirus vectors contain genetically engineered forms of adenovirus. The genetic organization of adenovirus is known to be a 36 kb, linear, double-stranded DNA virus, allowing replacement of large segments of adenoviral DNA with up to 7 kb of foreign sequence. Because adenoviral DNA can replicate in an episomal manner without potential genotoxicity, adenoviral infection of host cells, in contrast to retrovirus, does not result in chromosomal integration. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification.

[0066] Adenoviruses are particularly suitable for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis-receptors necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcription units separated by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the viral 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 host cell shutoff. Late gene products, including most of the viral 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 transcribed from this promoter contain a 5'-triple leader (TPL) sequence, making them preferred mRNAs for translation.

[0067] Other than the requirement that the adenoviral vector be replication-deficient, or at least conditionally-deficient, the nature of the adenoviral vector is not believed to be critical to the successful practice of certain embodiments disclosed herein. The adenovirus may be of any of the 42 different known serotypes or subgroups A through F. In certain embodiments, adenovirus type 5 of subgroup C is the preferred starting material for obtaining a conditionally replication-deficient adenoviral vector for use in certain embodiments, because considerable biochemical and genetic information is known about adenovirus type 5 and it is the human adenovirus that has historically been most used in constructs using adenoviruses as vectors.

[0068] As shown, 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 at a position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequence is not critical. A polynucleotide encoding a gene of interest may also be inserted in place of a deleted E3 region in an E3 replacement vector or in place of the E4 region when a helper cell line or helper virus complements the E4 deficiency.

[0069] Adeno-associated virus (AAV) is a parvovirus that was discovered as a contaminant of adenovirus strains. It is a ubiquitous virus (antibodies are present in 85% of the US human population) and has not been associated with any disease. It is also classified as a dependovirus because its replication is dependent on the presence of a helper virus, such as adenovirus. Various serotypes have been isolated, and the AAV-2 serotype is the best-characterized AAV, with a single linear DNA strand encapsidated within the capsid proteins VP1, VP2, and VP3 to form icosahedral virions measuring 20–24 nm in diameter.

[0070] AAV DNA is 4.7 kilobases long. It contains two open reading frames flanked by two ITRs. The AAV genome contains two essential genes: rep and cap. The rep gene encodes proteins responsible for 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 essential cis-components of AAV for chromosomal integration. Therefore, AAV can be used as a vector in which all viral coding sequences have been removed and replaced with a cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40 according to their map locations. Transcription from p5 and p19 leads to the production of rep proteins, and transcription from p40 produces capsid proteins.

[0071] AAV stands out for use within the present disclosure because of its excellent safety profile, because it rarely integrates into genomic DNA, and because its capsid and genome can be tailored to allow expression in selected cell populations. scAAV refers to self-complementary AAV. rAAV refers to recombinant adeno-associated virus.

[0072] Other viral vectors may also be used, such as vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus, which offer several attractive features for a variety of mammalian cells.

[0073] Retrovirus. Retroviruses are common tools for gene delivery. "Retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear, double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes necessary to produce new viral particles.

[0074] 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), and Rous sarcoma virus (RSV), and lentiviruses.

[0075] "Lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary viruses include HIV (human immunodeficiency virus; including HIV types 1 and 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis 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., HIV cis-acting sequence elements) may be used.

[0076] A "self-inactivating" (SIN) vector refers to a replication-deficient vector in which the right (3') LTR enhancer-promoter region (known as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because during viral replication, the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region; therefore, viral transcripts cannot be made without the U3 enhancer-promoter. In a further embodiment, the 3' LTR is modified such that the U5 region is replaced, for example, with a typical poly(A) sequence. It should be noted that modifications to the LTRs, such as modifications to the 3' LTR, modifications to the 5' LTR, or modifications to both the 3' and 5' LTRs, are also included in certain embodiments.

[0077] In certain embodiments, the viral vector contains a TAR element. The term "TAR" refers to a "transactivation response" gene element located in the R region of a lentivirus (e.g., HIV) LTR. This element interacts with the lentivirus transactivator (tat) gene element to increase viral replication. However, this element is not required in embodiments in which the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0078] "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping group (i.e., the beginning of transcription) and ends just before the beginning of the poly(A) tail. The R region is also defined by being flanked by the U3 and U5 regions. The R region is responsible for moving the nascent DNA from one end of the genome to the other during reverse transcription.

[0079] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various posttranscriptional regulatory elements increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and others (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector contains a posttranscriptional regulatory element such as a WPRE or HPRE. In certain embodiments, the vector lacks or does not contain a posttranscriptional regulatory element such as a WPRE or HPRE.

[0080] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcription can increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. The term "poly(A) site" or "poly(A) sequence" describes a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcription by RNA polymerase II. Polyadenylation sequences promote mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thus contributing to increased transcription efficiency. Certain embodiments may use BGHpA or SV40pA. In certain embodiments, preferred expression constructs contain termination elements. These elements contribute to increasing transcription levels and minimizing readthrough from the construct to other plasmid sequences.

[0081] Beyond the foregoing, a wide variety of suitable expression vector types are known to those of skill in the art. These can include commercially available expression vectors (e.g., plasmids containing one or more reporter genes and the regulatory elements necessary for expression of the reporter genes in cells) designed for common recombinant procedures. Many vectors are commercially available from, for example, Invitrogen, Stratagene, Clontech, etc., and are described in many accompanying manuals. In certain embodiments, suitable expression vectors include any plasmid, cosmid, or phage construct (e.g., the pUC or Bluescript plasmid series) capable of supporting expression of an encoded gene in mammalian cells.

[0082] Particular embodiments include:

[0083] [Table 1]

[0084] In certain embodiments, a viral vector having a capsid that crosses the blood-brain barrier (BBB) ​​is selected. In certain embodiments, the AAV is modified to contain a capsid that crosses the BBB. 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), and AAV-PPS (Chen et al., Nat Med. 2009;15:1215).

[0085] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, crosses the BBB after intravenous administration. It transduces large nodes of the central nervous system (CNS), thereby enabling minimally invasive treatment (Naso et al., BioDrugs. 2017;31(4):317), as described in connection with ongoing clinical trials for the treatment of superior mesenteric artery (SMA) syndrome with AveXis (AVXS-101, NCT03505099) and CLN3 gene-associated neuronal ceroid lipofuscinosis (NCT03770572). In certain embodiments, a representative AAV9 capsid protein sequence can include the AAV9VP1 capsid protein sequence (UniProt Accession No. Q6JC40, SEQ ID NO: 57).

[0086] AAVrh.10 was originally isolated from rhesus macaques and exhibits low serum soreness in humans when compared to other common serotypes used for gene delivery applications (Selot et al., Front Pharmacol. 2017;8:441), and is currently being evaluated in clinical trials LYS-SAF302, LYSOGENE, and NCT03612869.

[0087] Two variants isolated from a library of chimeric AAV vectors (AAVrh.10 replacing the AAV1 capsid domain), AAV1R6 and AAV1R7, retain the ability to cross the BBB and transduce the CNS, while showing significantly reduced liver and vascular endothelial transduction.

[0088] rAAVrh.8, also isolated from Reesis macaques, shows comprehensive transduction of glial and neuronal cell types in areas of clinical importance after peripheral administration, and also exhibits reduced peripheral tissue tropism compared to other vectors.

[0089] AAV-BR1 is an AAV2 variant that expresses the NRGTEWD ​​(SEQ ID NO: 53) epitope, isolated in an in vivo screening of a random AAV-display peptide library. It exhibits high specificity, with high transgene expression in the brain and minimal off-target affinity (including the liver) (Korbeline et al., EMBO Mol Med. 2016;8(6):609).

[0090] 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: 54), which potently transduces neurons in the enteric nervous system and peripheral sensory afferents, penetrating the spinal cord and brainstem.

[0091] 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: 55), which delivers genes throughout the CNS more efficiently than AAV9, transducing a large number of astrocytes and neurons across multiple CNS regions.

[0092] AAV-PPS is an AAV variant created by insertion of the DSPAHPS (SEQ ID NO: 56) epitope into the capsid of AAV2, and displays dramatically improved brain tropism relative to AAV2.

[0093] In certain embodiments, capsids are selected that, after administration (e.g., iv administration), result in brain-wide transduction of inhibitory cells in primates. In certain embodiments, capsids are selected that, after administration, result in widespread transduction of tissues and cell types affected by Scn1a deletion.

[0094] Expression constructs for administration. The vectors of the present disclosure (referred to herein as physiologically active components) may be formulated with a carrier suitable for administration to a human or animal subject. The physiologically active components in the compositions described herein may be prepared in neutral form, as free base, or as pharmaceutically acceptable salts.

[0095] Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as, for example, acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0096] Carriers for physiologically active ingredients include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, etc. The use of such carriers for physiologically active ingredients is known in the art. Except for conventional media or agents that are incompatible with the physiologically active ingredients, they may be used with the compositions described herein.

[0097] The phrase "pharmaceutically acceptable carrier" refers to a carrier that does not produce an allergic or similar untoward reaction when administered to humans, and in certain embodiments, when administered intravenously.

[0098] In certain embodiments, the compositions may be formulated for intravenous, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, oral, intraperitoneal, oral or nasal inhalation, or direct injection into one or more cells, tissues, or organs.

[0099] The composition may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres and / or nanoparticles.

[0100] The formation and use of liposomes are generally known to those skilled in the art. Liposomes have been developed to have improved serum stability and circulation half-life (see, e.g., U.S. Patent No. 5,741,516). Furthermore, various methods for the preparation of liposomes and liposome-like preparations as potential drug carriers have been described (see, e.g., U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868; and 5,795,587).

[0101] Liposomes are formed from phospholipids dispersed in aqueous media and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs), which generally have diameters between 25 nm and 4 μm). Sonication of MLVs results in the formation of small small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 angstroms, containing an aqueous solution in their cores.

[0102] Liposomes are similar to cell membranes and are widely suitable for both water-soluble and oil-soluble substances, ie, they can be entrapped both in the aqueous space and within the bilayer itself.

[0103] Liposomes can also be used for site-specific delivery of active agents by selectively modifying the liposome formulation. Because specific targeting is desirable, available methods for this purpose are established. For example, antibody binding domains can be attached to the liposome surface and used to direct the antibody and its drug contents to specific antigenic receptors located on the surface of specific cell types. Carbohydrate determinants (cell surface components of glycoproteins or glycolipids that play a role in cell-cell recognition, interaction, and adhesion) can also be used as recognition sites capable of directing liposomes to specific cell types.

[0104] In addition to the teachings of Couvreur et al. (FEBS Lett. 84(2):323-326, 1977; Crit Rev Ther Drug Carrier Syst. 5(1)1-20, 1988), the following information can be used in preparing liposome formulations: When dispersed in water, phospholipids can form various structures other than liposomes, depending on the lipid-to-water molar ratio. Liposomes at low ratios are the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but at elevated temperatures, they undergo a phase transition that significantly alters their permeability. The phase transition involves a change from a tight, ordered structure known as the gel state to a looser, less ordered structure known as the fluid state. This occurs at a characteristic phase transition temperature and increases permeability to ions, sugars, and drugs.

[0105] The ability to entrap solutes varies depending on the type of liposome. For example, MLVs are moderately efficient at entrapping solutes, while SUVs are very inefficient. While SUVs offer the advantages of uniformity and reproducibility of size distribution, a compromise between size and entrapment efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and dehydration and are 3–4 times more efficient than MLVs at entrapping solutes.

[0106] Alternatively, the present disclosure provides pharmaceutically acceptable nanocapsule formulations of the physiologically active components of the present disclosure. Nanocapsules generally 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 due to intracellular polymer overload, nanoparticles can be designed using in vivo degradable polymers. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure. Such particles can be prepared as described 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 US Pat. No. 5,145,684.

[0107] Injectable compositions may include sterile aqueous solutions or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable aqueous solutions or dispersions (U.S. Pat. No. 5,466,468). For delivery via injection, the form is sterile and fluid to the extent that it can be delivered by syringe. In certain embodiments, it contains a compound that is stable under the conditions of manufacture and storage and, optionally, preserved against the contaminating action of microorganisms, such as one or more bacteria and fungi. The carrier may be a solvent or dispersion medium, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), common mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersions, and / or by the use of surfactants. Prevention of the action of microorganisms may be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In various embodiments, the preparation may contain an isotonic agent, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. The injectable composition is suitably buffered if necessary, and the diluent is first rendered isotonic with sufficient saline and glucose.

[0108] Dispersions may also be prepared in glycols, liquid polyethylene glycols and mixtures thereof, and in oils. As indicated, under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0109] Sterile compositions can be prepared by incorporating the physiologically active ingredients in an appropriate amount of solvent with other optional ingredients (e.g., as listed above) and sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized physiologically active ingredients into a sterile vehicle containing a basic dispersion medium and other desired ingredients (e.g., as listed above). In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation can be vacuum drying and freeze-drying, which yield powders containing the physiologically active ingredients and any additional desired ingredients from a previously sterilized, filtered solution.

[0110] Oral compositions can be in liquid form, for example, solution, syrup or suspension, or can be presented as the preparation that is reconstituted before use with other suitable vehicle.Such liquid preparations can be prepared by conventional means using suspending agent (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fat); emulsifying agent (for example, lecithin or acacia); non-aqueous vehicle (for example, almond oil, oily ester or fractionated vegetable oils); and pharmaceutically acceptable additives such as preservative (for example, methyl or propyl-p-hydroxybenzoate or sorbic acid). The compositions may take the form of, for example, tablets or capsules prepared in a conventional manner with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); 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); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

[0111] Inhalable compositions may be delivered in the form of aerosol spray preparations from pressurized packs or nebulizers by using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mix of the compound and a suitable powder base (e.g., lactose or starch).

[0112] Compositions may also 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).

[0113] Supplementary active ingredients can also be incorporated into the compositions.

[0114] Typically, a composition will contain at least 0.1% or more of the physiologically active component, although the percentage of the physiologically active component can, of course, vary and may conveniently comprise 1 or 2% to 70% or 80% or more, or even between 0.5 and 99% by weight or volume of the total composition. Generally, the amount of physiologically active component in each physiologically useful composition can be adjusted for any given unit volume of the compound in a manner that provides a suitable dosage. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations. Accordingly, various compositions and dosages may be desirable.

[0115] In certain embodiments, for human administration, compositions must meet sterility, pyrogenicity, and certain safety and purity standards as required by the U.S. Food and Drug Administration (FDA) or other appropriate regulatory agency in other countries.

[0116] Methods of Use. In certain embodiments, a composition comprising a physiologically active component described herein is administered to a subject, resulting in selective expression of a protein or nucleotide sequence that rescues voltage-gated sodium channel function in inhibitory neurons in the subject. In certain embodiments, rescuing voltage-gated sodium channel function involves converting interneurons of the subject that do not have sufficient amounts and / or activity of Nav1.1 sodium channels into interneurons that express sufficient amounts and activity of exogenous voltage-gated sodium channels to restore neuronal function and prevent epileptic circuit activity.

[0117] In certain embodiments, rescued voltage-gated sodium channel function is evidenced by one or more of an increase in sodium channel current and / or an increase in excitability of inhibitory neurons engineered with physiologically active components. In certain embodiments, rescued voltage-gated sodium channel function is evidenced by one or more of an increase in sodium channel conductance and / or sodium channel influx in response to voltage depolarization of inhibitory neurons engineered with physiologically active components. The increase can be at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, or at least a 90% increase. Inhibitory neuron output can be measured using electrophysiological methods, such as multi-electrode arrays or patch clamps.

[0118] In certain embodiments, the inhibitory neuron is an inhibitory interneuron, a GABAergic neuron, a GABAergic interneuron, a pan-GABAergic neuron, or an inhibitory neuron in the hippocampus or cortex.

[0119] In certain embodiments, rescued voltage-gated sodium channel function is evidenced by an increase in sodium current-dependent rapid spiking in forebrain interneurons, e.g., using a mouse model. In certain embodiments, rescued voltage-gated sodium channel function is evidenced by a delay or suppression of temperature-induced seizing in a mouse model described herein.

[0120] Detailed embodiments include identifying a subject with reduced Nav1.1 sodium channel function in inhibitory neurons. Such a subject may be identified based on a diagnosis of a disorder associated with Nav1.1 sodium channel dysfunction, including epilepsy, SCN1A-associated seizure disorder, simple febrile seizures (FS), GEFS+, DS, refractory childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC), refractory partial seizures of infancy, myoclonic-astatic epilepsy, Lennox-Gastaut syndrome (LGS), and infantile spasms. Refractory seizures (also called "uncontrollable" or "intractable" seizures) are seizures that cannot be controlled to a satisfactory degree based on normal medical judgment with conventional treatment.

[0121] With regard to DS, specifically, although 80% of DS patients test positive for SCN1A gene mutations, the absence of an SCN1A mutation does not exclude a DS diagnosis. DS has been associated with mutations in the SCN1A (e.g., partial or complete deletion mutations, truncating mutations, and / or missense mutations, e.g., in the S4-S6 voltage domain or in the pore domain), 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 genes.

[0122] In certain embodiments, a subject in need of the treatments described herein may not experience diagnosable seizures but may exhibit subclinical discharges (referred to as a high rate of seizure-like activity when measured by electroencephalography (EEG)). Epilepsy syndromes associated with these seizure-like discharges include Landau-Kleffner syndrome and continuous spike-wave activity during slow-wave sleep.

[0123] In certain embodiments, the patient may have an intellectual and developmental disability (IDD), such as an autism spectrum disorder (ASD). In certain embodiments, the patient of the disclosed methods has epilepsy and an IDD or ASD disorder. Common IDD and ASD disorders that co-occur with seizures and epilepsy include Fragile X Syndrome (FXS), Rett Syndrome (RTT), Angelman Syndrome, Prader-Willi Syndrome, Palatocardiofacial Syndrome, Smith-Lemli-Opitz Syndrome, neuroligin mutations, and "interstitial neuropathies," which result from aristaless-related homeobox X-linked (ARX) and neuropilin 2 (NRP2) gene mutations.

[0124] The methods described herein are particularly useful for treating children and infants, and for treating disorders that develop during infancy or childhood. In certain embodiments, the patient of the disclosed methods is a newborn, infant, toddler, preschooler, school-age child, tween, or teen. In certain embodiments, the patient is 18 years of age or younger, 12 years of age or younger, 10 years of age or younger, 8 years of age or younger, 6 years of age or younger, 4 years of age or younger, 2 years of age or younger, or 1 year of age or younger. In certain embodiments, the patient is an adult over 18 years of age.

[0125] In certain embodiments, the methods reduce or prevent seizures or their symptoms in a patient in need thereof. In certain embodiments, the methods may reduce or prevent one or more different types of seizures. Ideally, the methods of the present disclosure result in complete prevention of seizures. However, the present disclosure also encompasses methods in which the incidence of seizures is reduced 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%.

[0126] Generally, seizures may include convulsions, repetitive movements, paresthesia, and combinations thereof. Seizures can be classified as focal seizures (also called partial seizures) and generalized seizures. Focal seizures occur on only one side of the brain, while generalized seizures occur on both sides of the brain. Specific types of focal seizures include simple focal seizures, complex focal seizures, and secondary generalized seizures. Simple focal seizures may be limited to or focused on a particular lobe (e.g., the temporal lobe, frontal lobe, parietal lobe, or occipital lobe). Complex focal seizures generally affect a larger portion of one hemisphere than simple focal seizures, but generally occur in the temporal or frontal lobe. If a focal seizure spreads from one side (hemisphere) of the brain to both sides, the seizure is called a secondary generalized seizure. Specific types of generalized seizures include absence seizures (also called petit mal), tonic seizures, atonic seizures, myoclonic seizures, tonic chronic seizures (also called grand mal), and chronic seizures.

[0127] In certain embodiments, the methods described herein may reduce the frequency of seizures, reduce the severity of seizures, change the type of seizures (e.g., from more severe to less severe), or a combination thereof, in a patient after treatment compared to no treatment (e.g., before treatment) or compared to treatment with an alternative conventional therapy.

[0128] Administration of the composition may be by any suitable route. For example, in certain embodiments, administration may include application to cells or tissue slices for research purposes related to Nav1.1 sodium channel dysfunction.

[0129] In certain embodiments, administration to a subject may be intravenous, retroorbital, intraocular, intravitreal, parenteral, subcutaneous, intracerebroventricular, intramuscular, intrathecal, intraspinal, oral, intraperitoneal, oral, nasal, or direct administration to a targeted site. Delivery may be achieved by needle or cannula, or by any other technique for ejecting a fluid substance. Methods of administration may include those described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515; and U.S. Pat. No. 5,399,363.

[0130] As is well known in the medical field, the dosage for any one subject will depend on many factors, including the subject's size, surface area, age, the particular compound being administered, sex, time and route of administration, general condition, and other drugs being administered concomitantly. Dosages of compounds of the present disclosure may vary, but in certain embodiments, the dose is approximately 10 times that of an expression construct of the present disclosure. 5 ~10 10 In certain embodiments, patients receiving intravenous, intraspinal, retroorbital, intraventricular, or intrathecal administration receive 10 copies of the expression construct. 6 ~10 22 It may be injected by copying.

[0131] A therapeutically effective amount includes an amount that is effective and / or provides therapeutic treatment.

[0132] An "effective amount" is the amount of a composition that produces a desired physiological change in a subject. Often, an effective amount is administered for research purposes. An effective amount of Nav1.1 of the present disclosure may produce a statistically significant effect in an animal model or in vitro assay for a disorder associated with sodium channel dysfunction.

[0133] "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of a disorder associated with Nav1.1 sodium channel dysfunction, and is administered to a subject for the purpose of reducing or eliminating the signs or symptoms of the disorder. Therapeutic treatment can reduce, control, or eliminate the presence or activity of the disorder, the etiology of the disorder, and / or the side effects of the disorder.

[0134] In certain embodiments, administration of a therapeutically effective amount of a composition of the present disclosure may be achieved in a single administration, e.g., a single administration of a sufficient number of expression constructs to provide a therapeutic benefit to the subject receiving the administration. Alternatively, in some situations, it may be desirable to provide multiple, sequential, or relatively short or long-term administrations of the composition.

[0135] For example, the number of expression constructs administered to a subject may be 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or more expression constructs / ml, or may be administered in a single dose or divided into two or more administrations as needed to achieve the desired physiological result. In certain embodiments, it may be desirable to administer two or more different expression constructs, alone or in combination with one or more other therapeutic drugs, to achieve the desired effect of a particular treatment regimen.

[0136] The dosage and timing of administration for a particular subject can be selected by the treating clinician, researcher, or veterinarian. In other words, the amount and time of administration of the composition and / or expression construct are within the skill of one of ordinary skill in the art having the benefit of the present teachings.

[0137] In certain embodiments, a treatment for a Nav1.1 sodium channelopathy may be combined with another treatment. For example, common conventional treatments for seizures and epilepsy include antiepileptic drugs and non-antiepileptic drug treatments such as low-carbohydrate diets (e.g., ketogenic diets, e.g., classical diets, medium-chain triglyceride (MCT) diets, modified Atkins diets, and low glycemic index therapy (LGIT)), intravenous immunoglobulins, steroids, elimination diets, vagus nerve stimulation, corticetomy, and multiple subpial transections.

[0138] 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.

[0139] Kits and Commercial Packages. Kits and commercial packages include the expression constructs described herein. The expression product may be isolated in certain embodiments. In certain embodiments, components of the expression product may be isolated from each other. In certain embodiments, the expression product may be in a vector, a viral vector, a cell, a tissue section or sample, and / or a transgenic animal. In certain embodiments, the animal is transgenic after administration of a composition comprising the expression construct. In certain embodiments, the transgenic animal contains a genetic modification that makes the animal suitable for use in an animal model of DS. For example, a transgenic animal, such as a mouse, may express Scn1a + / - Detailed methods for producing transgenic animals are described in U.S. Patent No. 4,736,866. The transgenic animals may be any non-human mammal or bird, but preferably include mice and non-human primates (NHPs). Also included are sheep, horses, cows, pigs, goats, dogs, cats, rabbits, chickens, and other rodents such as guinea pigs, hamsters, gerbils, rats, and ferrets.

[0140] Kit or commercial package embodiments also include instructions for use of the included composition, e.g., in research and / or for the treatment of disorders associated with Nav1.1 sodium channel dysfunction, such as epilepsy and / or DS. Such kits may further include one or more reagents, restriction enzymes, peptides, therapeutic agents, pharmaceutical compounds, or a means for delivery of the composition, such as a syringe, injectable, etc.

[0141] The following exemplary embodiments are included to illustrate specific embodiments of the present disclosure. Those of skill in the art should, in light of the present disclosure, recognize that many changes can be made to the specific embodiments disclosed herein and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0142] Exemplary embodiments. 1. An expression construct (e.g., an isolated polynucleotide) comprising: (i) a concatenated enhancer of SEQ ID NO: 2 or SEQ ID NO: 6; (ii) a promoter; and (iii) a coding sequence encoding a protein or nucleic acid that rescues voltage-gated sodium channel function in a cell or subject in need of rescue. 2. The expression construct of embodiment 1, wherein the concatemer comprises 3X concatemers of SEQ ID NO:2 or SEQ ID NO:6. 3. The expression construct of embodiment 1, wherein the concatemer comprises SEQ ID NO:2 and SEQ ID NO:6 in tandem. 4. The expression construct of embodiment 3, wherein the concatemers comprise SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:2-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:2; or SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:6. 5. An expression construct (e.g., an isolated polynucleotide) comprising: (i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8, (ii) a promoter; and (iii) a coding sequence encoding a protein or nucleic acid that rescues voltage-gated sodium channel function in a cell or subject in need of rescue. 6. The expression construct of any of embodiments 1-5, wherein the coding sequence comprises or encodes NavSheP-D60N, NavBp, NavMs, 3xHA-NavSheP-D60N, 3xHA-NavBp, 3xHA-NavMs, or His-NavMs. 7. The expression construct of any of embodiments 1-6, wherein the coding sequence comprises or encodes human SCN1A, mouse Scn1a, human SCN1A-3xHA, and / or mouse Scn1a-3Xha. 8. The expression construct of any of embodiments 1 to 7, wherein the coding sequence comprises or encodes an artificial transcription factor that increases expression of endogenous SCN1A, and the artificial transcription factor comprises a targeting DNA-binding domain linked to a transcription activation domain. 9. The expression construct of any of embodiments 1-8, wherein the coding sequence comprises or encodes an antisense RNA molecule that increases splicing or prevents negative regulation of a microRNA. 10. The expression construct of any of embodiments 1-9, wherein the coding sequence comprises or encodes a nucleotide sequence that upregulates SCN1A expression (e.g., SEQ ID NOs: 66-163). 11. The expression construct of any of embodiments 1-10, wherein the coding sequence comprises or encodes one or more segments of SCN1A that, upon expression, assemble into full-length SCN1A. 12. The expression construct of embodiment 11, comprising the SCN1A coding sequences of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. 13. The expression construct of embodiment 11, wherein the SCN1A segment comprises SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65. 14. The expression construct of any of embodiments 1-13, wherein the promoter comprises minBglobin or minCMV. 15. The expression construct of any of embodiments 1-14, wherein the expression construct is in an adeno-associated virus (AAV) vector. 16. The expression construct of any of embodiments 1-15, wherein the expression construct comprises a coding sequence for a reporter protein. 17. The expression construct of embodiment 16, wherein the reporter protein comprises a fluorescent reporter protein. 18. The expression construct of any of embodiments 1-17, wherein the expression construct comprises or encodes a skipping element. 19. The expression construct of embodiment 18, wherein the skipping element comprises a 2A peptide or an internal ribosome entry site (IRES). 20. The expression construct of embodiment 19, wherein the 2A peptide comprises T2A, P2A, E2A, and / or F2A. 21. The expression construct of any of embodiments 1-20, wherein the construct comprises elements of CN1367, CN1244, CN1389, CN1390, CN1180, CN1203, CN1498, CN1499, CN1500, CN2001, CN2002, CN2003, CN1504 and CN1512, CN2004 and CN2005, CN2006 and CN2007, CN2008 and CN2009, or CN2026, CN2027, CN2028 and CN2029. 22. The expression construct of any of embodiments 1-21, wherein the construct comprises SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and / or SEQ ID NO:52. 23. The expression construct of any of embodiments 1-22, wherein the construct comprises SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, or CN2026, CN2027, CN2028 and CN2029. 24. The expression construct of any of embodiments 15-23, wherein the AAV vector is bound to a capsid that crosses the blood-brain barrier. 25. The expression construct of embodiment 24, wherein the capsid comprises PHP.Eb. 26. The expression construct of embodiment 24 or 25, wherein the capsid comprises SEQ ID NO: 53. 27. The expression construct of any of embodiments 24-26, wherein the capsid comprises an AAV9 capsid having an insert of SEQ ID NO: 54 or SEQ ID NO: 55. 28. The expression construct of any of embodiments 24-27, wherein the capsid comprises an AAV2 capsid having a sequence number 56 insert. 29. A composition comprising the expression construct of any of embodiments 1 to 28. 30. A cell comprising the expression construct of any of embodiments 1 to 28. 31. A non-human animal comprising the expression construct of any of embodiments 1 to 28. 32. A kit comprising the expression construct of any of embodiments 1 to 28. 33. A method of rescuing voltage-gated sodium channel function in a defective cell in need of rescue, comprising administering to the cell a therapeutically effective amount of the composition of embodiment 29. 34. A method of rescuing voltage-gated sodium channel function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of embodiment 29. 35. The method of embodiment 34, wherein the subject in need of rescue results from a diagnosis of epilepsy, an SCN1A-associated seizure disorder, simple febrile seizures (FS), generalized epilepsy febrile seizures plus (GEFS+), Dravet syndrome (DS), refractory childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC), refractory partial infantile seizures, myoclonic astatic epilepsy, Lennox-Gastaut syndrome (LGS), or infantile spasms. 36. The method of embodiment 34 or 35, wherein the subject is a pediatric patient. 37. The method of any of embodiments 34-36, wherein the subject is under 4 years of age. 38. Subjects are transgenic Scn1a mice undergoing a temperature-induced febrile seizure test. + / - The method of embodiment 34, 35, or 37, wherein the subject is a mouse. 39. The method of any of embodiments 34-38, wherein the composition is administered intravenously. 40. The method of any of embodiments 34-39, wherein the composition is administered intrathecally into the cerebrospinal fluid via a cannula into the lateral ventricle or cisterna magna or lumbar space, or the foramen magnum. 41. (a) a non-naturally occurring enhancer sequence; and (b) a nucleic acid or nucleotide sequence encoding a protein that rescues voltage-gated sodium channel function; an expression construct comprising: The enhancer sequence is (i) consists of the sequence of SEQ ID NO: 3; (ii) contains two or more copies of SEQ ID NO:2 or SEQ ID NO:6 in tandem (e.g., SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:6-SEQ ID NO:2; SEQ ID NO:2-SEQ ID NO:2-SEQ ID NO:6; SEQ ID NO:6-SEQ ID NO:2-SEQ ID NO:2; or SEQ ID NO:2-SEQ ID NO:6-SEQ ID NO:6; SEQ ID NO:6-SEQ ID NO:6); (iii) comprises a sequence that has at least 90% sequence identity to SEQ ID NO:3 and maintains interneuron-specific enhancer function; or (iv) the complement of a sequence capable of hybridizing to the sequence of (i), (ii), or (iii), An expression construct (e.g., an isolated polynucleotide) that, after administration to a sample or subject, selectively promotes transcription of a nucleic acid in inhibitory neurons. 42. The expression construct of embodiment 41, wherein the nucleic acid comprises or encodes NavSheP-D60N, NavBp, NavMs, 3xHA-NavSheP-D60N, 3xHA-NavBp, 3xHA-NavMs, or His-NavMs. 43. The expression construct of embodiment 41 or 42, wherein the nucleic acid comprises or encodes human SCN1A, mouse Scn1a, human SCN1A-3xHA, and / or mouse Scn1a-3Xha. 44. The expression construct of any of embodiments 41 to 43, wherein the nucleic acid comprises or encodes an artificial transcription factor that increases expression of endogenous SCN1A, wherein the artificial transcription factor comprises a targeting DNA binding domain linked to a transcription activation domain. 45. The expression construct of any of embodiments 41 to 44, wherein the nucleic acid comprises or encodes an antisense RNA molecule that increases splicing or prevents negative regulation of a microRNA. 46. ​​The expression construct of any of embodiments 41-45, wherein the nucleic acid comprises or encodes a nucleotide sequence (e.g., SEQ ID NOs: 66-163) that upregulates SCN1A expression. 47. The expression construct of any of embodiments 41-46, wherein the nucleic acid comprises or encodes one or more segments of SCN1A that, after expression, assemble into full-length SCN1A. 48. The expression construct of embodiment 47, comprising a nucleic acid comprising the SCN1A coding sequence of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. 49. The expression construct of embodiment 47, wherein the SCN1A segment comprises SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65. 50. The expression construct of any of embodiments 41 to 49, comprising a promoter comprising minBglobin or minCMV. 51. The expression construct of any of embodiments 41-50, wherein the expression construct is in an adeno-associated virus (AAV) vector. 52. The expression construct of any of embodiments 41-51, wherein the expression construct comprises a nucleic acid encoding a reporter protein. 53. The expression construct of embodiment 52, wherein the reporter protein comprises a fluorescent reporter protein. 54. The expression construct of any of embodiments 41-53, wherein the expression construct comprises or encodes a skipping element. 55. The expression construct of embodiment 54, wherein the skipping element comprises a 2A peptide or an internal ribosome entry site (IRES). 56. The expression construct of embodiment 55, wherein the 2A peptide comprises T2A, P2A, E2A, and / or F2A. 57. The expression construct of any of embodiments 41-56, wherein the construct comprises elements of CN1367, CN1244, CN1389, CN1390, CN1180, CN1203, CN1498, CN1499, CN1500, CN2001, CN2002, CN2003, CN1504 and CN1512, CN2004 and CN2005, CN2006 and CN2007, or CN2008 and CN2009. 58. The expression construct of any of embodiments 41-57, wherein the construct comprises SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and / or SEQ ID NO:52. 59. The expression construct of any of embodiments 41-58, wherein the construct comprises SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, or SEQ ID NO:51 and SEQ ID NO:52. 60. The expression construct of any of embodiments 51-59, wherein the AAV vector is bound to a capsid that crosses the blood-brain barrier. 61. The expression construct of embodiment 60, wherein the capsid comprises PHP.Eb. 62. The expression construct of embodiment 60 or 61, wherein the capsid comprises SEQ ID NO: 53. 63. The expression construct of any of embodiments 60-62, wherein the capsid comprises an AAV9 capsid having an insert of SEQ ID NO: 54 or SEQ ID NO: 55. 64. The expression construct of any of embodiments 60-63, wherein the capsid comprises an AAV2 capsid having an insert of SEQ ID NO: 56. 65. A composition comprising the expression construct of any of embodiments 41 to 64. 66. A cell comprising the expression construct of any of embodiments 41 to 64. 67. A non-human animal comprising the expression construct of any of embodiments 41 to 64. 68. A kit comprising the expression construct of any of embodiments 41 to 64. 69. A method of rescuing voltage-gated sodium channel function in a defective cell in need of rescue, comprising administering to the cell a therapeutically effective amount of the composition of embodiment 65. 70. A method of rescuing voltage-gated sodium channel function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of embodiment 65. 71. The method of embodiment 70, wherein the subject in need of rescue results from a diagnosis of epilepsy, an SCN1A-associated seizure disorder, simple febrile seizures (FS), generalized epilepsy febrile seizures plus (GEFS+), Dravet syndrome (DS), refractory childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC), refractory partial infantile seizures, myoclonic astatic epilepsy, Lennox-Gastaut syndrome (LGS), or infantile spasms. 72. The method of any of embodiments 70 or 71, wherein the subject is a pediatric patient. 73. The method of any of embodiments 70-72, wherein the subject is under 4 years of age. 74. Subjects are transgenic Scn1a mice undergoing a temperature-induced febrile seizure test. + / - The method of any of embodiments 70, 71, or 73, wherein the mouse is a mouse. 75. The method of any of embodiments 70-74, wherein the composition is administered intravenously. 76. The method of any of embodiments 70-75, wherein the composition is administered intrathecally into the cerebrospinal fluid via a cannula into the lateral ventricle or cisterna magna or lumbar space, or the foramen magnum. 77. A vector system comprising two AAV vectors, wherein the first vector encodes the N-terminal portion of a protein that rescues voltage-gated sodium channel activity in a cell in need of rescue, and the second vector encodes the C-terminal portion of said protein, wherein the portions of the gene encoded by the two vectors overlap to provide regions of homology for homologous recombination to generate the full-length Nav1.1 protein, and the first vector comprises a promoter and an enhancer consisting of SEQ ID NO:3 but does not comprise a termination signal or polyA signal, and the second vector comprises a termination signal and polyA signal but does not comprise a promoter or enhancer. 78. The vector system of embodiment 77, wherein the protein comprises human SCN1A. 79. The vector system of embodiment 77 or 78, wherein the homology region is 75 to 1000 base pairs. 80. The vector system of embodiment 77 or 78, wherein the homology region is 550 to 650 base pairs. 81. The vector system of any of embodiments 77-80, wherein the vector selectively expresses the protein in inhibitory neurons. 82. The vector system of any of embodiments 77 to 81, wherein the homology region further comprises an intron element with a splicing donor site in the first vector and a splicing acceptor site in the second vector, such that trans-splicing across the homology region drives more efficient full-length protein reconstitution after recombination. 83. The vector system of any of embodiments 77-82, wherein the vector system comprises elements of CN1504, CN1512, CN2004, CN2005, CN2006, CN2007, CN2008, and / or CN2009. 84. The vector system of any of embodiments 77-83, wherein the vector system comprises elements of CN1504 and CN1512, CN2004 and CN2005, CN2006 and CN2007, or CN2008 and CN2009. 85. The vector system of any of embodiments 77 to 84, wherein the vector system comprises SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, or SEQ ID NO: 51 and SEQ ID NO: 52. 86. A viral vector comprising SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. 87. An expression construct comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8 and a coding sequence encoding an artificial transcription factor comprising a specific DNA binding domain linked to a general transcription activation domain, wherein the artificial transcription factor is an artificial transcription factor that increases expression of an endogenous functional copy of SCN1A (or other SCN_A family gene). 88. An expression construct comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8 and a coding sequence encoding an artificial transcription factor comprising a specific DNA binding domain linked to a general transcription activation domain, wherein the artificial transcription factor is an artificial transcription factor that increases expression of an endogenous functional copy of SCN1A (or other SCN_A family gene). 89. An expression construct comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8 and a coding sequence encoding an antisense RNA molecule that increases splicing of SCN1A expression and / or prevents negative regulation of microRNAs. 90. A series of expression constructs comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and / or SEQ ID NO:8, wherein the series of expression constructs deliver the hSCN1A protein as two or four subunit ORFs delivered by two or four separate vectors. 91. An isolated polynucleotide comprising an enhancer comprising the sequence of SEQ ID NO: 3, and further comprising a heterologous inducible promoter and a gene or nucleic acid sequence encoding a protein that selectively rescues Nav1.1 channel function in inhibitory neurons, wherein the enhancer and the heterologous inducible promoter are operably linked to the gene. 92. A vector comprising the isolated polynucleotide of embodiment 91. 93. The vector of embodiment 92, wherein the vector is a viral vector. 94. The vector of embodiment 93, wherein the viral vector is an AAV viral vector. 95. A human or non-human cell comprising the isolated polynucleotide of embodiment 91. 96. A series of vectors comprising any of the vectors of embodiments 92 to 94, which selectively drive expression of a protein or nucleic acid sequence that rescues Nav1.1 channel function in inhibitory neurons, wherein the second vector comprises an enhancer selected from SEQ ID NO: 1, 2, 4, 5, 6, 7 or 8. 97. A method for selectively driving the expression of a protein or nucleic acid sequence that rescues Nav1.1 channel function in inhibitory neurons, comprising the steps of: (1) providing a vector of any of embodiments 92 to 94; (2) producing a transgenic mouse using the vector; and (3) detecting the rescued Nav1.1 channel function in the transgenic mouse. 98. The method of embodiment 97, wherein the rescued Nav1.1 channel function is in an inhibitory neuron. 99. A series of coding sequences including the coding sequences of the hSCN1A_fragments in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. 100. A series of vectors including CN2026, CN2027, CN2028, and CN2029. [Example]

[0143] Example 1. Referring to Figure 1A, live human temporal cortex brain tissue was excised during neurosurgery to remove epileptic foci. The tissue was dissected and sliced ​​into 350-micron-thick tissue slices and cultured on a semipermeable membrane using a semi-synthetic medium described by Ting et al. (Scientific Reports 8(1):8407, 2018). On the first day of culture, the slices were infected with purified virus CN1180 / DJ (which labels inhibitory neurons) by directly applying the virus to the brain slices. Seven days after infection, SYFP2 expression in infected cells was significantly increased. + The electrophysiology of the cells (visible in the green channel) was assessed by patch clamp recording. Recorded cells were back-stained with Alexa 594 dye (visible in the red channel) and post-hoc visualization of their cell bodies and morphology was performed.

[0144] With reference to Figure 1B, patch clamp recordings were performed in multiplex with SYFP2 + Cells and SYFP2 - The analysis was performed on cells. Backstained morphology was tabulated as pyramidal (indicating excitatory neurons) or non-pyramidal (indicating inhibitory neurons). This analysis was performed using SYFP2 + showed that the cells were mostly characteristic of inhibitory neurons.

[0145] Regarding Figure 1C, multiplexed SYFP2 + Cells and SYFP2 - The electrophysiological characteristics of the cells were analyzed. - Compared to cells, SYFP2 + The cells showed shorter action potential (AP) half-widths, greater firing frequencies, faster depolarization rates, and faster AP firing rates (FI slopes) in response to injected current. These matrices all expressed SYFP2. + We showed that the cells were rapidly spiking interneurons.

[0146] Regarding Figure 1D, after patch clamp electrophysiology was performed, tissues were fixed and immunostained with anti-GFP and anti-parvalbumin antibodies. + The cells express parvalbumin + This indicated that these cells have a molecular identity consistent with rapidly spiking interneurons.

[0147] With reference to Figure 3, Gad2-T2A-nls-mCherry (Peron et al., Neuron 86:783-799, 2015) mice were injected retro-orbitally with 1E11 genome copies of the virus CN1244 / PHP.eB. Three weeks later, brains were harvested, sliced ​​into 350 micron-thick sections, and imaged for SYFP2 and mCherry expression to visualize virus-expressing cells and total interneurons, respectively.

[0148] For Figures 4A and 4B, adult wild-type mice were injected retro-orbitally with 1E+11 genome copies of the indicated viruses. Animals were maintained for 3-4 weeks, then euthanized, and brains were removed and sectioned, followed by live-tissue epifluorescence imaging of native fluorescence. Exposure times were adjusted to allow direct comparison of transgene expression levels. The first three panels are 500 ms exposures for each of the indicated constructs, and the fourth panel is a shorter (50 ms) exposure of CN1390.

[0149] For Figure 5, cortical / hippocampal brain slice cultures were prepared from P5-10 Gad2-IRES-Cre heterozygous animals; Ai75 heterozygous animals. These animals had Cre-mediated activation of the nuclear-tagged tdTomato transgene in Gad2-expressing cells, resulting in bright red nuclear fluorescence in inhibitory neurons. After 1 hour of culture, CN1390 virus suspension was pipetted onto the slice surface to transduce brain cell types. At 10 DIV / 10 DPI, native fluorescence was imaged in the green and red channels with a Nikon inverted microscope.

[0150] With reference to Figures 6A-6E, human ex vivo neocortical brain slice cultures were prepared from live neurosurgical specimens (Ting et al., Scientific Reports 8(1):8407, 2018). After 1 h of culture, CN1390 virus suspension was pipetted onto the slices to transduce brain cell types. At 1, 3, and 6 days per day (DIV / DPI), native SYFP2 fluorescence was imaged using adapted exposure times on a Nikon microscope. Figures 6A-6D illustrate rapid viral genetic labeling of human neocortical interneurons for targeted patch clamp recording and analysis. At various culture times, slices were removed for terminal patch clamp recording analysis, and the firing characteristics of labeled neurons were analyzed. Functional analysis of the firing patterns and electrical characteristics of human neocortical interneurons by patch clamp recording was feasible as early as 40 h after infection with the CN1390 eB virus.

[0151] With reference to Figure 9, the top vector contains the enhancer and promoter (minCMV) elements of SEQ ID NO: 3 and drives expression in all inhibitory neurons (including both Pvalb+ and Pvalb- inhibitory neurons). The driven transgene contains SYFP2 linked to a P2A tag to the N-terminal region of human SCN1A, which contains 604 bp of homology to the C-terminal region contained in the bottom vector. The bottom vector also contains a C-terminal 3xHA tag and 3'UTR regulatory sequences (WPRE3 and polyA site).

[0152] Both AAV viral vectors were packaged within PHP.eB capsids and delivered intravenously to a single C57Bl / 6 mouse. 21 days later, mouse brains were harvested, fixed, and processed for immunofluorescence with anti-GFP (targeting SYFP2), anti-HA, and anti-Pvalb antibodies to detect transgene-expressing cells and their overlap with Pvalb inhibitory neurons (cells of particular importance in DS syndrome). Many inhibitory neurons, including both Pvalb inhibitory neurons and Pvalb inhibitory neurons, were highly labeled with the N-terminal SYFP2 tag. Some of these SYFP2 cells were also able to express HA, indicating that the intact SCN1A protein product was expressed within these GFP HA cells (arrows). Vertical arrows indicate Pvalb interneurons expressing human SCN1A, and horizontal arrows indicate Pvalb interneurons expressing human SCN1A. These SCN1A-expressing cells are found throughout the forebrain (in both the neocortex and hippocampus [here the dentate gyrus]), including brain regions known to be important in most epilepsies and shown to be dysfunctional in DS.

[0153] Example 2. Dravet syndrome (DS) is a drug-resistant and life-threatening form of epilepsy. It typically begins in the first year of life with febrile or temperature-induced seizures that progress to generalized clonic, tonic-clonic, and unilateral seizures. These seizures are often resistant to current antiepileptic drugs, which are the first-line treatment for this syndrome; complete seizure control is typically not achieved. As the disease progresses, most affected children suffer from comorbid symptoms, including developmental delay, intellectual disability, impaired motor control and coordination, autistic behavior, and sleep disorders, and many die before reaching maturity.

[0154] Heterozygous loss-of-function mutations in SCN1A, the gene encoding the pore-forming subunit of the voltage-gated sodium channel Nav1.1, are the most common cause of DS, affecting approximately 1 in 16,000 newborns.

[0155] A mouse model generated by knockout of Scn1a recapitulates some of the key phenotypic features of this epilepsy, including infantile (P21) epilepsy onset, high susceptibility to febrile seizures, ataxia, spontaneous seizures, sleep disorders, autistic behavior, and premature death. Seizures and several complications result from interneuron damage in this mouse model.

[0156] This mouse model was used to investigate the efficacy of a novel viral vector for DS. The virus was delivered by retro-orbital injection using an insulin syringe, and its ability to suppress seizures was evaluated using a febrile seizure test. In this test, the internal temperature of the mouse was slowly increased using a temperature controller and heat lamp until a seizure occurred or until a temperature of 42.5°C was reached. The temperature at seizure onset in treated and control mice was compared to determine the effectiveness of the intervention. In further studies, the efficacy of treatment against spontaneous seizures and early mortality were assessed using video and electroencephalography monitoring.

[0157] The viral vector is a novel AAV viral vector designated CN1500. This viral vector is a recombinant AAV that expresses the transgene SYFP2-P2A-NavSheP-D60N to rescue defects in the voltage-gated sodium channel Nav1.1. NavSheP-D60N is a bacterially derived modified voltage-gated sodium channel that has been modified to improve kinetics and expression in mammalian cells. Transgene expression levels are increased by the addition of a WPRE3 element, and transcription is terminated by the bovine growth hormone polyadenylation sequence. Transgene expression, mediated by the 3xhi56iCore synthetic enhancer (SEQ ID NO: 3) directly 5' of the CMV minimal promoter, is high and restricted to inhibitory cells in forebrain structures, including the cortex and hippocampus. Furthermore, the therapeutic transgene NavSheP-D60N is tagged with an HA epitope tag to assess correct protein localization.

[0158] To test the efficacy of therapeutic AAV viral vectors, we used the CN1500 package with the PHP.eB serotype. + / - Cohorts of mice were divided into 2x10 11 The mice were either injected with 1000 mg of AAV or left uninjected. AAV was introduced intravenously using a retro-orbital route of delivery. Two weeks after virus administration, animals from the treatment and control groups were evaluated for susceptibility to febrile seizures. As previously described, febrile seizures were assessed by steadily increasing the mice's body temperature by 0.5°C every 2 minutes under a heat lamp and measuring the mice's internal temperature using a rectal probe. The body temperature of mice that experienced seizures was recorded.

[0159] The novel therapeutic vector CN1500 was highly expressed in GABAergic cells in both the cortex and hippocampus of mice, but not in Scn1a cells that had undergone febrile seizures. + / - The mean body temperature of the mice also increased from 38.7° C. to 41° C. These data indicate that CN1500 can substantially rescue Scn1a deficiency.

[0160] References for Example 2 include: Catterall et al. (2010) The Journal of physiology 588:1849-1859; Cheah et al. (2012) Proceedings of the National Academy of Sciences of the United States of America 109:14646-14651; Kalume (2013) Respir Physiol Neurobiol.189(2):324-8; Kalume et al., (2007) J Neurosci 27:11065-11074; Kalume et al., (2013) The Journal of clinical investigation 123:1798-1808; Oakley et al., (2009) Proceedings of the National Academy of Sciences of the United States of America 106:3994-3999.

[0161] Sequence description The nucleic acids described herein are depicted using standard abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, although the complementary strand is considered to be included in the embodiments, where appropriate.

[0162] SEQ ID NO: 1: nucleic acid sequence of human (h)DLXI56i enhancer; SEQ ID NO:2: nucleic acid sequence of hI56icore enhancer; SEQ ID NO:3: nucleic acid sequence of 3xhI56iCore enhancer; SEQ ID NO: 4: nucleic acid sequence of mouse DLXI56i enhancer; SEQ ID NO: 5: nucleic acid sequence of zebrafish DLXI56i enhancer; SEQ ID NO: 6: nucleic acid sequence of zebrafish I56icore; SEQ ID NO: 7: nucleic acid sequence of zebrafish 3xI56icore; SEQ ID NO: 8: hDLXI12b enhancer; SEQ ID NO: 9: NavSheP-D60N, codon optimized, with N-terminal 3xHA tag; SEQ ID NO: 10: NavSheP endogenous sequence; SEQ ID NO: 11: NavBp, endogenous sequence; SEQ ID NO: 12: NavBp, codon-optimized, with N-terminal 3xHA tag; SEQ ID NO: 13: NavMs, endogenous sequence; SEQ ID NO: 14: NavMs, codon-optimized, with N-terminal 3xHA tag and linker; SEQ ID NO: 15: NavMs, codon-optimized, with N-terminal His tag and linker; SEQ ID NO: 16: human SCN1A; SEQ ID NO: 17: SYFP2; SEQ ID NO: 18: P2AEn coding sequence; SEQ ID NO: 19: WPRE3; SEQ ID NO: 20: BGHpA; SEQ ID NO: 21: N-terminal 3XHA tag (protein); SEQ ID NO: 22: N-terminal 3XHA tag (DNA); SEQ ID NO: 23: N-terminus of hSCN1A2 partial expression system; SEQ ID NO: 24: C-terminus of hSCN1A2 partial expression system, containing the C-terminal 3XHA sequence; SEQ ID NO: 25: 604 bp homology region of hSCN1 A term and C term, which can be used in a two-part expression system; SEQ ID NO:26: P2A translation from CN1498; SEQ ID NO: 27: T2A; SEQ ID NO:28: E2A; SEQ ID NO:29:F2A; SEQ ID NO: 30: MinBglobin; SEQ ID NO: 31: minCMV; SEQ ID NO: 32: AAV9PHP.eB capsid replacement sequence; SEQ ID NO: 33: CN1367-portion between L-ITR and R-ITR: positions 142-2984; SEQ ID NO: 34: CN1500-portion between L-ITR and R-ITR: positions 142-2976; SEQ ID NO: 35: CN1498-portion between L-ITR and R-ITR: positions 142-2943; SEQ ID NO: 36: CN1499-portion between L-ITR and R-ITR: positions 142-2946; SEQ ID NO: 37: CN1244-portion between L-ITR and R-ITR: positions 142-2042; SEQ ID NO: 38: CN1389-portion between L-ITR and R-ITR: positions 142-1897; SEQ ID NO: 39: CN1390-portion between L-ITR and R-ITR: positions 142-1660; SEQ ID NO: 40: CN1203-portion between L-ITR and R-ITR: positions 183-2052; SEQ ID NO: 41: CN1180-portion between L-ITR and R-ITR: positions 183-1891; SEQ ID NO: 42: CN2001-portion between L-ITR and R-ITR: positions 142-2023; SEQ ID NO: 43: CN2002-portion between L-ITR and R-ITR: positions 142-1993; SEQ ID NO: 44: CN2003-portion between L-ITR and R-ITR: positions 142-2056; SEQ ID NO: 45: CN1504-portion between L-ITR and R-ITR: positions 142-4489; SEQ ID NO: 46: CN1512-portion between L-ITR and R-ITR: positions 142-4165; SEQ ID NO: 47: CN2004-portion between L-ITR and R-ITR: positions 142-3792; SEQ ID NO: 48: CN2005-portion between L-ITR and R-ITR: positions 142-4160; SEQ ID NO: 49: CN2006-portion between L-ITR and R-ITR: positions 142-4790; SEQ ID NO: 50: CN2007-portion between L-ITR and R-ITR: positions 142-4671; SEQ ID NO: 51: CN2008-portion between L-ITR and R-ITR: positions 142-3995; SEQ ID NO: 52: CN2009-portion between L-ITR and R-ITR: positions 142-4525; SEQ ID NO: 53: Epitope isolated during in vivo screening of a random AAV display peptide library; SEQ ID NO: 54: sequence of AAV-PHP.S7-mer; SEQ ID NO: 55: sequence of AAV-PHP.B7-mer; SEQ ID NO: 56: sequence of AAV-PPS 7-mer; SEQ ID NO: 57: AAV9VP1 capsid protein sequence (UniProt accession number Q6JC40); SEQ ID NO: 58: CN2026-rAAV-3xhI56i(core)-minBG-hSCN1A_fragment1-WPRE3-BGHpA; SEQ ID NO: 59: CN2027-rAAV-3xhI56i(core)-minBG-hSCN1A_fragment2-WPRE3-BGHpA; SEQ ID NO: 60: CN2028-rAAV-3xhI56i(core)-minBG-hSCN1A_fragment3-WPRE3-BGHpA; SEQ ID NO: 61: CN2029-rAAV-3xhI56i(core)-minBG-hSCN1A_fragment4-WPRE3-BGHpA; SEQ ID NO: 62:>hSCN1A_fragment1_protein sequence; SEQ ID NO: 63: hSCN1A_fragment2_protein sequence; SEQ ID NO: 64: hSCN1A_fragment3_protein sequence; SEQ ID NO: 65: hSCN1A_fragment4_protein sequence; SEQ ID NOs: 66-163: Nucleotide sequences that result in upregulation of SCNA1.

[0163] Variants of the sequences disclosed and referenced herein are also included. Guidance on determining which amino acid residues may be substituted, inserted, or deleted without destroying biological activity can be found in the DNASTAR® method, which is well known in the art. TMConservative amino acid changes can be found using computer programs such as software. Preferably, the amino acid changes in the protein variants disclosed herein are substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of a family of amino acids that are related in their side chains.

[0164] In a peptide or protein, suitable conservative amino acid substitutions are known to those of skill in the art and are generally made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families as follows: 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 (Arg), lysine (Lys), and histidine (His); Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0165] In making such changes, the hydropathic index of the amino acid is taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are: 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); Glutamate (-3.5); Gln (-3.5); Aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0166] Certain amino acids can be substituted with other amino acids having similar hydropathic indexes and scores, still resulting in proteins with similar biological activity, i.e., still resulting in biologically functionally equivalent proteins. Such changes are preferably substituted with amino acids having hydropathic indexes of ±2, more preferably within ±1, and even more preferably within ±0.5. It is also understood in the art that substitution of similar amino acids can be effectively performed on the basis of hydropathicity.

[0167] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Aspartate (+3.0±1); Glutamate (+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 understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still maintain biological equivalence, particularly immunological equivalence. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are more preferred, and those within ±0.5 are even more preferred.

[0168] As outlined above, amino acid substitutions can 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.

[0169] As noted elsewhere, variants of a gene sequence may include codon-optimized variants, sequence polymorphisms, splicing variants, and / or mutations that do not affect the function of the encoded protein to a statistically significant degree.

[0170] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, and gene sequences disclosed herein.

[0171] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be easily calculated by known methods, including but not limited to those described in the following: 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 give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignment can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty = 10, gap length penalty = 10).Suitable programs also include the GCG suite of programs (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). Within the context of this disclosure, when sequence analysis software is used, it is understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set of parameter values ​​that are initially loaded with the software when the software is first initialized.

[0172] Variants also include nucleic acids that hybridize to the sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include: overnight incubation at 42°C in a solution containing 50% formamide, 5X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared salmon sperm DNA, followed by washing the filter with 0.1X SSC at 50°C. Altering the stringency of hybridization and signal detection is achieved initially through manipulation of formamide concentration (lower percentages of formamide result in lower stringency), salt conditions, or temperature. For example, moderately high hybridization conditions include overnight incubation at 37°C in a solution containing 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaHPO; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml salmon sperm blocking DNA; followed by a wash at 50°C with 1X SSPE and 0.1% SDS. Additionally, to achieve lower stringency, stringent hybridization may be followed by a wash with a higher salt concentration (e.g., 5X SSC). Variations on the above conditions may be achieved through the inclusion and / or exclusion of alternative blocking reagents used to suppress background in hybridization experiments. Representative blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

[0173] In certain embodiments, references to gene editing systems, such as CRISPR and Cas-9 (also known as Csnl and Csxl2) mentioned above, should be interpreted as incorporating further options and developments, as will be understood by those skilled in the art. For example, any gene editing system that allows precise genome recognition and binding may be utilized. When a nuclease is used for targeted genome binding without cleavage and editing, the nuclease may lack cleavage functionality.

[0174] In relation to the use of Cas-9, many other options for Cas proteins are available and suitable for use. Cas-9 itself refers to one or more catalytic domains of Cas9 proteins derived from bacteria such as Corynebacterium, Satellella, Legionella, Treponema, Filifacter, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Spirochete, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, Cas9 is a fusion protein, e.g., where the two catalytic domains are derived from different bacterial species. Additional exemplary Cas nucleases that can be used to bind DNA include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, C2c3, C2c2 and C2c1, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.

[0175] Cpf1 nucleases may offer additional flexibility in target site selection, particularly by means of a short three-base pair recognition sequence (TTN) known as a protospacer adjacent motif, or PAM. Particular embodiments may utilize engineered Cpf1s. For example, US 2018 / 0030425 describes engineered Cpf1 nucleases from the Lachnospiraceae bacteria ND2006 and Acidaminococcus sp. BV3L6 that have been engineered to alter and improve target specificity.

[0176] Other Cpf1 variants include Cpf1 homologs and orthologs of the Cpf1 polypeptides disclosed in Zetsche et al. (2015) Cell 163:759-771 and the Cpf1 polypeptides disclosed in US2016 / 0208243. Other engineered Cpf1 variants are known to those of skill in the art and are included within the scope of the present disclosure (see, e.g., WO / 2017 / 184768).

[0177] For further information regarding CRISPR-Cas systems and their components, see US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US89996 41 and related applications; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO201 4 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089 354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and related applications.

[0178] Zinc finger nucleases (ZFNs) can be synthesized by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. Detailed embodiments described herein may utilize zinc fingers that bind to specific DNA sequences. The DNA-binding domain contains three to six zinc finger proteins, similar to those found in transcription factors. The DNA cleavage domain includes, for example, the catalytic domain of FokI endonuclease. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for sites on either side of the target site cleavage sequence. The FokI cleavage site cleaves within a 5-6 base pair spacer sequence, splitting into two opposing halves.

[0179] For further information regarding ZFNs and ZFNs useful in the teachings of this disclosure, see, e.g., U.S. Patent Nos. 534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; No. 6,479,626; and U.S. Patent Application Publication Nos. 2003 / 0232410 and 2009 / 0203140, as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161, 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 609-1614 (1985).

[0180] A detailed embodiment may use transcription activator-like effector nucleases (TALENs) as gene editing agents. TALENs are engineered to bind target gene sequences and cleave DNA at the target sequence. TALEN TALEs are DNA-binding proteins secreted by Xanthomonas bacteria. The DNA-binding domain of TALEs contains highly conserved 33 or 34 amino acid repeats, with divergent residues at the 12th and 13th positions of each repeat. These two positions, called repeat variable dimers (RVDs), show strong correlation with specific nucleotide recognition. Therefore, targeting specificity can be improved by modifying amino acids in the RVDs and incorporating non-conventional amino acids.

[0181] Examples of DNA cleavage domains that can be used in TALEN fusions are wild-type and variant FokI endonucleases. For more information regarding TALENs, see U.S. Patent Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853, as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55; Beurdeley et al., Nat Commun, 2013, 4:1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. al. Science 326, 1509-1512 (2009); and Moscow, & Bogdanove, Science 326, 1501 (2009).

[0182] A detailed embodiment can use MegaTAL as a gene editing agent. MegaTAL has a rare-cleaving single-chain nuclease structure, in which TALE is fused with the DNA cleavage domain of meganuclease. Meganuclease, also known as homing endonuclease, is a single peptide chain that has both DNA recognition function and nuclease function in the same domain. In contrast to TALEN, megaTAL only requires the delivery of a single peptide chain for functional activity.

[0183] Examples of meganucleases include I-Scel, I-Scell, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ceul, I-CeuAIIP, I-Crel, I-CrepsblP, I-CrepsbllP, I-CrepsbIIIP, I-CrepsbIVP, I-Tlil, I-Ppol, PI-PspI, F-Scel, F-Scell, F-Suvl, F-Tevl, F-TevII, I-Amal, I-Anil, I-Chul, I-Cmoel, I-Cpal, I-CpaII, I-Csml, I-Cvul, I-CvuAIP, I-Ddil, I-DdiII, I-Dirl, I-Dmol, I-Hmul, I-HmuII, I-HsNIP, I-Llal, I-Msol, I-Naal, I-Nanl, I-NcIIP, I-NgrIP, I-Nitl, I-Njal, I-Nsp236IP, I-Pakl, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrlP, 1-PobIP, I-Porl, I-PorIIP, I-PbpIP, I-SpBetaIP, I-Scal, I-SexIP, 1-SneIP, I-Spoml, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-Tevl, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinlP, 1-ZbiIP, PI-MtuI, PI-MtuHIPPI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, Pl-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-Tful, PI-TfuII, PI-Thyl, PI-Tlil, and PI-THII.

[0184] Detailed embodiments described herein may utilize gene editing systems to insert expression constructs into targeted genomic safe harbors. Methods for identifying genomic safe harbor sites are described in Sadelain et al., Nature Reviews (2012); 12:51-58; and Papapetrou et al., Nat Biotechnol. (2011) January; 29(1):73-8.

[0185] In certain embodiments, an expression construct refers to an isolated polynucleotide sequence that does not contain any elements, portions, or nucleotides not described in connection with the construct.

[0186] As will be understood by those skilled in the art, each embodiment disclosed herein may comprise, consist essentially of, or consist of the specifically mentioned elements, steps, ingredients, or components. Accordingly, the terms "include" or "including" should be interpreted to list: "comprise," "consist of," or "consist essentially of." The transitional terms "comprise" or "comprises" mean including, but are not limited to, and allow for the inclusion of unspecified elements, steps, ingredients, or components, even in significant amounts. The transitional phrase "consisting of" excludes all unspecified elements, steps, ingredients, or components. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the specified elements, steps, ingredients, or components and those that do not materially affect the embodiment. Substances that substantially significantly reduce the ability of the vector to overcome febrile seizures in Scn1a+ / - mice follow the protocol of Example 2 and FIG.

[0187] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties contemplated to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, all numerical parameters should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarity is needed, the term "about" has the meaning reasonably ascribed to the term and when used in conjunction with a stated numerical value or range by one of ordinary skill in the art means somewhat more or somewhat less than the stated numerical value or range, and means within ±20% of the stated numerical value; ±19% of the stated numerical value; ±18% of the stated numerical value; ±17% of the stated numerical value; ±16% of the stated numerical value; ±15% of the stated numerical value; ±14% of the stated numerical value; ±13% of the stated numerical value; ±12% of the stated numerical value; ±11% of the stated numerical value; ±10% of the stated numerical value; ±9% of the stated numerical value; ±8% of the stated numerical value; ±7% of the stated numerical value; ±6% of the stated numerical value; ±5% of the stated numerical value; ±4% of the stated numerical value; ±3% of the stated numerical value; ±2% of the stated numerical value; or ±1% of the stated numerical value.

[0188] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value necessarily contains certain errors resulting from the standard deviation found in their respective testing measurements.

[0189] Unless otherwise indicated or clearly contradicted by context, the terms "a," "an," "the," and similar referents used in the context of describing the present invention (particularly in the context of the claims that follow) are intended to cover both the singular and the plural. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") presented herein is intended only to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0190] The categorization of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Members of each group may be referred to, claimed individually, or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be incorporated into, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or exclusion occurs, the specification is deemed to include the group as modified to fulfill all Markush group descriptions used in the claims.

[0191] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, modifications of these described embodiments will be apparent to those skilled in the art in light of the foregoing description. The inventors anticipate that such modifications will be employed by those skilled in the art as appropriate, and the inventors intend the invention to be practiced unless otherwise stated herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, in accordance with applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0192] Additionally, numerous references are made throughout this specification to patents, publications, journal articles, and other writings (the "references" herein), each of which is individually incorporated by reference in its entirety into this specification for the teachings to which it refers.

[0193] Finally, it is understood that the embodiments of the invention disclosed herein are for purposes of illustrating the invention. Other modifications that may be employed are 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 of the invention. Accordingly, the invention is not limited to that precisely as shown and described.

[0194] The details set forth herein are for illustrative purposes and are presented solely to illustrate a discussion of preferred embodiments of the present invention, and to provide what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, there is no intention to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention, and the present description, together with the figures and / or examples, will make clear to those skilled in the art how some forms of the present invention may be actually implemented.

[0195] The definitions and explanations used in this disclosure are meant and intended to govern any future constructions unless clearly and unambiguously modified according to the examples, or if the application of meanings results in any structural or substantive contradiction. In the event of a conflict or substantive contradiction in the construction of terms, the definitions shall be taken from Webster's Dictionary, Third Edition (Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004)), which is known to those skilled in the art.

Claims

1. (a) an enhancer, (b) a promoter, and (c) a nucleic acid encoding a protein that rescues voltage-gated sodium channel function; 1. An expression construct comprising: the nucleic acid is selected from NavSheP-D60N, NavBp, NavMs, 3xHA-NavSheP-D60N, 3xHA-NavBp, 3xHA-NavMs, His-NavMs, human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA; The enhancer may comprise: (i) comprises two or more copies of SEQ ID NO:2 arranged in tandem; or (ii) comprising two or more copies of a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 2 arranged in tandem and maintaining interneuron-specific enhancer function; An expression construct wherein, after administration to a sample or a subject, the enhancer promotes transcription of the nucleic acid in inhibitory neurons.

2. The expression construct of claim 1, wherein the enhancer comprises three copies of sequence number 2 arranged in tandem, or three copies of a sequence having at least 95% sequence identity with the sequence of sequence number 2 arranged in tandem and maintaining interneuron-specific enhancer function.

3. 3. The expression construct of claim 2, wherein the enhancer comprises the sequence of SEQ ID NO:3 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO:

3.

4. The expression construct of claim 1 , wherein the promoter comprises minBglobin or minCMV.

5. the expression construct is in an adeno-associated virus (AAV) vector; wherein the nucleic acid encoding a protein that rescues voltage-gated sodium channel function comprises human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA, or when the nucleic acid encoding a protein that rescues voltage-gated sodium channel function is selected from human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA, the protein is expressed using at least a first AAV vector and a second AAV vector; the first AAV vector comprises: (I-i) enhancer, (I-ii) a promoter, and (I-iii) Nucleic acids encoding the N-terminal portions and regions of human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA but without a termination signal or polyA signal, the second AAV vector comprises: (I-iv) A nucleic acid encoding the C-terminal portion and region of human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA, when the nucleic acid defined in (I-iii) encodes the N-terminal portion and region of human SCN1A, mouse Scn1a, human SCN1A-3xHA, or mouse Scn1a-3xHA, respectively; (I-v) a termination signal, and (I-vi) Poly A signal but no promoter or enhancer, the portions of the nucleic acid encoded by the first AAV vector and the second AAV vector overlap to provide regions of homology for homologous recombination to generate a full-length Nav1.1 protein; 2. The expression construct of claim 1.

6. 2. The expression construct of claim 1, wherein the expression construct comprises or encodes a T2A peptide, a P2A peptide, an E2A peptide, and / or an F2A peptide, or an internal ribosome entry site (IRES).

7. The expression constructs are: hI56i-minBglobin-His-NavMs-P2A-SYFP2-WPRE3-BGHpA, 3xhI56iCore-minCMV-SYFP2-P2A-3xHA-NavBp-WPRE3-BGHpA, 3xhI56iCore-minCMV-SYFP2-P2A-3xHA-NavMs-WPRE3 -BGHpA, 3xhI56iCore-minCMV-SYFP2-P2A-3xHA-NavSheP-D60N-WPRE3-BGHpA, 3xhI56iCore- minBG-NavBp-WPRE3-BGHpA, 3xhI56iCore-minBG-NavMs-WPRE3-BGHpA, 3xhI56iCore-minBG- NavSheP-D60N-WPRE3-BGHpA, 3xhI56iCore-minCMV-SYFP2-P2A-hSCN1A-Nterm and hSCN1A-Cterm-3xHA-WPRE3-BGHpA, 3xhI56iCore-minBG-hSCN1AFrontEnd and hSCN1ABackEnd-WPRE3-BGHpA, 3xhI56iCore-minBG-SYFP2ns-P2 2. The expression construct of claim 1, comprising the elements A-hSCN1AFrontEnd-IntronBridge and IntronBridge-hSCN1ABackEnd-3xHA-WPRE3-BGHpA, or 3xhI56iCore-minBG-hSCN1AFrontEnd-IntronBridge and IntronBridge-hSCN1ABackEnd-WPRE3-BGHpA.

8. 2. The expression construct of claim 1, wherein the expression construct comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:

52.

9. The expression construct of claim 5 , wherein the AAV vector is associated with a capsid that crosses the blood-brain barrier.

10. A composition for rescuing voltage-gated sodium channel function in a subject or defective cell in need of rescue of voltage-gated sodium channel function, comprising a therapeutically effective amount of an expression construct described in claim 1.

11. 11. The composition of claim 10, wherein the subject is in need of the rescue due to a diagnosis of epilepsy, an SCN1A-associated seizure disorder, simple febrile seizures (FS), generalized epilepsy febrile seizures plus (GEFS+), Dravet syndrome (DS), refractory childhood epilepsy with generalized tonic-clonic seizures (ICE-GTC), refractory partial seizures of infancy, myoclonic astatic epilepsy, Lennox-Gastaut syndrome (LGS), or infantile spasms.

12. The composition of claim 10 , wherein the subject is a pediatric patient.

13. The composition of claim 10, wherein the subject is a transgenic Scn1a+ / - mouse undergoing a temperature-induced febrile seizure test.

14. 11. The composition of claim 10, wherein the composition is administered intravenously or intrathecally into the cerebrospinal fluid via a cannula into the lateral ventricle or cisterna magna or lumbar space, or the foramen magnum.

15. a vector system comprising two AAV vectors, the first vector encoding the N-terminal portion of a protein that rescues voltage-gated sodium channel activity in a cell in need of rescue, and the second vector encoding the C-terminal portion of the protein, wherein the portions of the gene encoded by the two vectors overlap to provide regions of homology for homologous recombination to generate the full-length Navl.l protein; the first vector comprises a promoter and an enhancer but does not comprise a termination signal or a polyA signal; The enhancer (i) comprises two or more copies of a sequence having at least 95% sequence identity to the sequence of SEQ ID NO:2, or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:3, arranged in tandem; and (ii) maintains interneuron-specific enhancer function. the second vector contains a termination signal and a polyA signal but does not contain a promoter or enhancer; Vector system.

16. 16. The vector system of claim 15, wherein the enhancer comprises three copies of a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:

2.

17. 17. The vector system of claim 16, wherein the enhancer comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:

3.

18. The vector system of claim 15 , wherein the vector expresses a protein in an inhibitory neuron.

19. 16. The vector system of claim 15, wherein the homology regions further comprise an intron element with a splicing donor site in the first vector and a splicing acceptor site in the second vector, such that trans-splicing across the homology regions drives more efficient full-length protein reconstitution after recombination.

20. 16. The vector system of claim 15, wherein the vector system comprises a sequence having at least 95% sequence identity with the sequences of SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, or SEQ ID NO:51 and SEQ ID NO:

52.

21. A viral vector comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.

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