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WO2025038955A9 - Systèmes et procédés pour améliorer la sécurité d'une thérapie génique médiée par aav à intéine divisée - Google Patents

Systèmes et procédés pour améliorer la sécurité d'une thérapie génique médiée par aav à intéine divisée Download PDF

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WO2025038955A9
WO2025038955A9 PCT/US2024/042744 US2024042744W WO2025038955A9 WO 2025038955 A9 WO2025038955 A9 WO 2025038955A9 US 2024042744 W US2024042744 W US 2024042744W WO 2025038955 A9 WO2025038955 A9 WO 2025038955A9
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polynucleotide sequence
scn1a
intein
seq
expression construct
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WO2025038955A1 (fr
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Franck KALUME
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Seattle Childrens Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • adeno-associated virus AAV
  • AAV adeno-associated virus
  • PTS split intein-mediated protein trans-splicing
  • a large protein coding sequence is split into two or more parts, each at least tagged on one end (or, for internal parts of the split sequence, flanked) by sequences that encode split-inteins, which are independently cloned in two or more AAV vectors.
  • split-inteins are expressed as two independent polypeptides (N-intein and C-intein) at the extremities of the host polypeptides (N-polypeptide and C-polypeptide) and remain inactive until encountering their complementary partner.
  • the reconstituted intein excises itself from the host protein while mediating ligation of the N- and C-polypeptides via a peptide bond, in a traceless manner.
  • AAV intein vectors encode components (e.g., excised inteins) of nonmammalian origin that could elicit immune or toxic responses in target cells and/or raise regulatory concerns for clinical translation.
  • AAV intein vectors are the delivery for expression and reconstitution of large protein molecules to supplement or salvage the function of endogenously deficient or mutated protein molecules due to one or more diseases or disorders.
  • Epilepsy is a neurological disorder that occurs when the brain presents an enduring predisposition to generate two or more epileptic seizures.
  • An epileptic seizure is a temporary disruption of brain function due to abnormal excessive or synchronous neuronal activity. Its manifestation may include periods of unusual behavior, sensations and sometimes loss of consciousness.
  • Dravet Syndrome (DS) particularly is a rare and catastrophic form of intractable epilepsy that begins in infancy. Initially, the patient experiences prolonged seizures.
  • the alpha subunit protein When the alpha subunit protein is expressed by a cell, it is able to form a pore in the cell membrane that conducts Na+ in a voltage-dependent way, even if beta subunits or other known modulating proteins are not expressed. When accessory proteins assemble with ⁇ subunits, the resulting complex can display altered voltage dependence and cellular localization. [0009] Therefore, it is an object of the present invention to provide a trans-splicing system with enhanced safety or reduced immunogenicity by removing byproducts. [0010] It is another object of the present invention to improve the safety or trans- splicing system for use in safe delivery of coding sequence of a sodium channel protein in the treatment of Dravet Syndrome or epilepsy disorders.
  • a system includes: (a) a first expression construct comprising: a first portion of a polynucleotide sequence of a gene encoding the sodium channel alpha subunit, and a polynucleotide sequence encoding an N-fragment of a split intein (N-intein), at the 3’ end relative to the first portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit; (b) a second expression construct comprising: a second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit, and a polynucleotide sequence encoding a C-fragment of the split intein (C-intein), at the 5’ end relative to the second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit; and (c) a third expression construct comprising a polynucleotide sequence encoding a degron.
  • a system includes: (a) a first expression construct comprising: a first portion of a polynucleotide sequence of a gene encoding the sodium channel alpha subunit, and a polynucleotide sequence encoding an N-fragment of a split intein (N- intein), at the 3’ end relative to the first portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit; (b) a second expression construct comprising: a second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit, and a polynucleotide sequence encoding a C-fragment of the split intein (C-intein), at the 5’ end relative to the second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit; wherein the first expression construct, the second expression construct, or both further comprises a polynucleotide sequence en
  • a system includes the first expression construct, the second expression, either one or both further comprising the polynucleotide sequence encoding the degron, and a third expression construct coding for a same or different degron.
  • a system for expression one or more coding sequences includes (a) a first expression construct coding for a first fusion protein comprising a first segment of a sodium channel alpha subunit and an N-fragment of a split intein, wherein the first segment of the sodium channel alpha subunit is at the N-terminus relative to the N- fragment of the split intein; (b) a second expression construct coding for a second fusion protein comprising a C-fragment of the split intein and a second segment of the sodium channel alpha subunit, wherein the second segment of the sodium channel alpha subunit is at the C-terminus relative to the C-fragment of the split intein; and (c) a polynucleotide sequence encoding a degron, wherein the polynucleotide sequence encoding the degron is in a third expression construct different from the first or the second expression constructs OR is within the first and/or the second expression constructs.
  • the first fusion protein and the second fusion protein are spliced together, thereby joining the first segment and the second segment of the sodium channel alpha subunit and joining the N-fragment and the C-fragment of the split intein.
  • the system comprising the first, second, and third expression constructs is co-transduced to a cell for expression of the first fusion protein, the second fusion protein, and the degron in the cell.
  • the split intein is one having fast splicing rate (e.g., half-lives within 5 min), such as consensus fast intein.
  • the degron is a polypeptide having no more than 30 amino-acid residues in length.
  • the split intein in the system is consensus fast intein
  • the degron in the system is one having 9-26 or no more than 30 amino-acid residues in length.
  • the splicing to joining the first and second segments of the sodium channel alpha subunit takes places faster than degron-mediated degradation, resulting in the joined segments (preferably a full) sodium channel alpha subunit locating in a cell membrane, whereas joined intein is degraded via degron.
  • the first and/or the second expression construct further comprises an enhancer sequence, configured for targeted expression within a targeted cell type.
  • the expression construct(s) further comprises a promoter sequence.
  • the first and/or the second expression construct further comprises an intron having a polynucleotide sequence of SEQ ID NO:107. In some embodiments, the first and/or the second expression construct further comprises the enhancer sequence, the promoter, and the intron.
  • the gene encoding the sodium channel alpha subunit is selected from the group consisting of SCN1A ⁇ SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, SCN11A, and SCN7A.
  • the sodium channel alpha subunit comprises sodium channel protein type 1 subunit alpha
  • the gene encoding the sodium channel alpha subunit comprises SCN1A.
  • the sodium channel alpha subunit comprises human sodium channel protein type 1 subunit alpha isoform 2. In some embodiments, the sodium channel alpha subunit comprises a variant of human sodium channel protein type 1 subunit alpha isoform 2 having an amino acid substitution of A1056T, wherein amino acid residue numbering is according to NCBI accession number NP_001340878.1.
  • the first expression construct comprises the polynucleotide sequence encoding the degron
  • the first expression construct comprises from 5’ to 3’: the first portion of the polynucleotide sequence of the SCN1A – the polynucleotide sequence encoding the N-intein – the polynucleotide sequence encoding the degron.
  • the degron has an amino acid sequence of RPGSTSPFAPSATDLPSMPEPALTSR (SEQ ID NO:91) or ACKNWFSSLSHFVIHL (SEQ ID NO:92).
  • the first expression construct comprises the polynucleotide sequence encoding the degron, and the first expression construct comprises from 5’ to 3’: the first portion of the polynucleotide sequence of the SCN1A – the polynucleotide sequence encoding the degron – the polynucleotide sequence encoding the N- intein.
  • the second expression construct comprises the polynucleotide sequence encoding the degron, and when expressed, the degron is two or more amino acid residues at the N-terminus relative to a protein product encoded by the second portion of the polynucleotide sequence of the SCN1A.
  • the degron has an amino acid sequence of MSCAQESITSLYKKAGSENLYFQ (SEQ ID NO:88), MSCAQES (SEQ ID NO:90), or GSLIIFIIL (SEQ ID NO:93).
  • the second expression construct comprises the polynucleotide sequence encoding the degron, and the second expression construct comprises from 5’ to 3’: a first portion of the polynucleotide sequence encoding the C-intein – the polynucleotide sequence encoding the degron – a second portion of the polynucleotide sequence encoding the C-intein – the second portion of the polynucleotide sequence of the SCN1A, wherein when expression, a protein product of the first portion of the polynucleotide sequence encoding the C-intein and a protein product of the second portion of the polynucleotide sequence encoding the C-intein together form the C-intein
  • a protein product of the first portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit and a protein product of the second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit are linked, via a peptide bond between the C-terminus of the first portion’s protein product and the N-terminus of the second portion’s protein product, to reconstitute the sodium channel alpha subunit.
  • the degron has an amino acid sequence selected from the group consisting of: MSCAQESITSLYKKAGSENLYFQ (SEQ ID NO:88), MSCAQES (SEQ ID NO:90), RPGSTSPFAPSATDLPSMPEPALTSR (SEQ ID NO:91), ACKNWFSSLSHFVIHL (SEQ ID NO:92), and GSLIIFIIL (SEQ ID NO:93).
  • the breakpoint of the first and second segment of the sodium channel alpha subunit is at a place wherein the first residue of the C-terminus segment (fragment) is Cys, Ser, or Thr.
  • the first and the second portions of the polynucleotide sequence of the SCN1A encode residues 1-1049 and residues 1050-1998 of sodium channel protein type 1 subunit alpha isoform 2 (or variants containing A1056T substitution), respectively.
  • the first and the second portions of the polynucleotide sequence of the SCN1A encode residues 1-956 and residues 957-1998 of the sodium channel protein type 1 subunit alpha isoform 2 (or variants containing A1056T substitution), respectively.
  • the first and the second portions of the polynucleotide sequence of the SCN1A encode residues 1-947 and residues 948-1998 of the sodium channel protein type 1 subunit alpha isoform 2 (or variants containing A1056T substitution), respectively.
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm1049 (SEQ ID NO: 59)
  • the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm949 (SEQ ID NO: 60).
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm956 (SEQ ID NO: 61), and the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm1042 (SEQ ID NO: 62).
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm947 (SEQ ID NO: 63), and the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm1051 (SEQ ID NO: 64).
  • SEQ ID NO: 64 polynucleotide sequence of hSCN1A-CO-Cterm1051
  • the split intein comprises consensus fast intein (Cfa);
  • the degron is a polypeptide being 5-30 amino-acid residues in length or preferably 9-26 amino- acid residues in length; and a polypeptide product encoded by the second portion of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit starts with a cystein, serine, or threonine residue.
  • the first segment and the second segment of the sodium channel alpha subunits are even or about even sized; or the lengths of segments are not more than 20%, 30%, 40%, or 50% different.
  • the polynucleotide sequence encoding the N-intein comprises a polynucleotide sequence of Cfa-N (SEQ ID NO:57), and the polynucleotide sequence encoding the C-intein comprises a polynucleotide sequence of Cfa-C (SEQ ID NO:58).
  • the first expression construct and the second expression construct independently further comprise the promoter sequence selected from a minBglobin promoter having a polynucleotide sequence of SEQ ID NO:3, an hSyn1 promoter having a polynucleotide sequence of SEQ ID NO:52, or a CMV promoter having a polynucleotide sequence of SEQ ID NO:53; optionally a shortened hSyn1 promoter having a polynucleotide sequence of SEQ ID NO: 54.
  • the first expression construct and the second expression construct independently further comprise the minBglobin promoter having a polynucleotide sequence of SEQ ID NO:3.
  • the enhancer sequence is configured for targeted expression of the first, the second, or both portions, respectively, of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit within a targeted central nervous system cell type.
  • the targeted central nervous system cell type is GABAergic neuron, glutamatergic neuron, or both cell types.
  • the targeted central nervous system cell type is GABAergic interneuron.
  • the enhancer sequence is set forth in SEQ ID NO: 2 (DLX2.0).
  • the enhancer sequence has a concatemerized core having a polynucleotide sequence of SEQ ID NO:1.
  • the enhancer sequence is a concatemerized repeat (2, 3, 4, 5, 6, 7, 8, 9, 10, or more contiguous repeats) of a polynucleotide sequence of SEQ ID NO:1.
  • an enhancer sequence is set forth in SEQ ID NO: 55 (eHGT_078h), or the targeted central nervous system cell type comprises a glutamatergic neuron.
  • the first expression construct, the second expression construct, or both independently further comprise a miRNA binding site sequence, configured for targeted expression of the first, the second, or both portions, respectively, of the polynucleotide sequence of the gene encoding the sodium channel alpha subunit within a selected central nervous system cell type.
  • the miRNA binding site sequence is set forth in SEQ ID NO: 56 (4x2C miRNA binding site) or SEQ ID NO:87 (8x2C miRNA binding site), or the selected central nervous system cell type comprises a pan- GABAergic neuron.
  • An artificial expression construct is also provided, which includes the first, the second and/or the third expression construct of a system disclosed herein, wherein each expression construct is associated with a capsid that crosses the blood brain barrier.
  • a capsid that crosses the blood brain barrier comprises PHP.eB.
  • a capsid that crosses the blood brain barrier comprises AAV-BR1.
  • a capsid that crosses the blood brain barrier comprises AAV-PHP.S.
  • a capsid that crosses the blood brain barrier comprises AAV-PHP.B. In some embodiments, a capsid that crosses the blood brain barrier comprises AAV-PPS.
  • an administrable composition which includes one or more artificial expression constructs disclosed herein, preferably in association with a capsid that crosses blood brain barrier; and a pharmaceutically acceptable excipient.
  • a transgenic cell is also provided comprising a system of one or more expression constructs disclosed herein. In some embodiments, the transgenic cell comprises a GABAergic neuron, or more specifically GABAergic interneuron. In some embodiments, the transgenic cell comprises a glutamatergic neuron.
  • Methods are also provided for rescuing voltage-gated sodium channel function within a targeted population of cells, the method comprising: co-administering a therapeutically effective amount of the two or more expression constructs of a system disclosed herein to a sample or subject comprising the targeted population of cells, and inducing expression of the expression constructs to reconstitute a sodium channel alpha subunit, thereby rescuing voltage-gated sodium channel function within the targeted population of cells.
  • Methods are also provided for administering a system of expression constructs to a subject in need thereof, the method comprising administering a therapeutically effective amount of a system disclosed herein, or co-administering two or more expression constructs of a system disclosed herein, to a sample or subject comprising the targeted population of cells, and inducing expression of the expression constructs to reconstitute a sodium channel alpha subunit, thereby rescuing voltage-gated sodium channel function within the targeted population of cells.
  • the subject has a sodium channelopathies, optionally comprising Dravet syndrome, myoclonic seizures, myoclonic astatic epilepsy (MAE), intractable childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epilepsy and febrile seizures plus (GEFS+), migrating partial seizures of infancy, Lennox-Gastaut syndrome, or West syndrome.
  • the subject is a pediatric patient having Dravet syndrome.
  • FIG. 1A A functional split-intein design of SCN1A that reconstitutes functional Na V1.1 activity.
  • FIG. 1A Design of split-intein fusion protein halves of SCN1A. We inserted the breakpoint and Cfa-N and Cfa-C split-intein peptides just before the native Cys1050 with no additional amino acids. After joining, the Cfa-N and Cfa-C intein fragments self-excise and yield scarless reconstituted SCN1A.
  • HA and FLAG epitopes are inserted at the N- and C-termini of the N- and C-terminal halves for detection.
  • FIG. 1B Cloning SCN1A split-intein fusion protein halves into CMV-driven plasmid vectors for testing functionality in cell lines, as well as IRES2-SYFP2 and IRES2-mScarlet transfection reporters.
  • FIG. 1C Reconstitution of full-length SCN1A after co-transfection of split-intein fusion protein halves into HEK-293 cells. We analyzed whole cell protein preparations by western blot for HA epitope tag after transfection.
  • Anti-tubulin is a loading control.
  • FIG.1D Exemplary currents evoked in HEK293 cells transfected with full- length SCN1A (SCN1A-FL, green), a combination of SCN1A-Ntm and SCN1A-Ctm (SCN1A-N+C, orange), SCN1A-Ntm only (SCN1A-N, blue) and SCN1A-Ctm only (SCN1A- C, blue), in response to a family of step depolarizations from a holding potential of -120 mV to 40 mV, in 5 mV increments.
  • FIG.1F Voltage-dependent gating properties of WT full-length human SCN1A (SCN1A-FL) and reconstituted SCN1A channels formed by co-expression of N-terminal SCN1A-Cfa intein (SCN1A-N) and Cfa intein-C- terminal SCN1A (SCN1A-C) plasmid constructs, acutely expressed in HEK293 cells and characterized by whole-cell patch-clamp recordings.
  • FIG. 2A Cell class-specific delivery of SCN1A to telencephalic GABAergic interneurons using optimized enhancer DLX2.0.
  • FIG. 2A Recombinant AAV2/PHP.eB vectors for delivery of DLX2.0-split-intein-SCN1A.
  • HA- and FLAG-expressing cells can be observed when either half is delivered alone or together. Layer 2/3 of VISp is shown at P20.
  • FIG. 2D Representative stitched fluorescence image of biodistribution of HA and FLAG epitopes in scattered telencephalic neurons. Expression is pseudo-colored black.
  • FIG.2E Representative stitched fluorescence image of HA and FLAG epitopes, and Gad67+ neurons in VISp. In panels D-E we show expression at P47.
  • FIG. 2F High specificity and completeness of expression within Gad67+ neurons in multiple telencephalic regions across multiple animals. We counted cells that express both HA and FLAG epitopes in visual VISp, MO, and HPF.
  • FIG.3A Recovery of mortality and epileptic symptoms in DS model mice with DLX2.0-split intein-SCN1A.
  • FIG.3A Experimental timeline to rescue of epileptic symptoms in DS model mice.
  • the untreated control groups in Fig. 3B-EE represents the same set of untreated animals as that shown in Fig. 7B, 7D-7F.
  • FIG. 3C-3E Protection from heat-induced seizures in DS model mice after treatment with DLX2.0-SCN1A AAVs.
  • FIG.3E DS model mice treated with DLX2.0 N+C SCN1A AAVs exhibit significantly fewer MC events during thermal challenge assay than untreated mice or mice treated with empty or single-part vector controls (** unpaired t-test p ⁇ 0.001 at each indicated timepoint, for comparison to Untreated and Empty/single part negative control animals).
  • Figures 4A-4F Recovery of spontaneous epileptic symptoms in DS model mice with DLX2.0-SCN1A AAVs.
  • FIG.4A-4B Interictal spike reduction in DS model mice.
  • FIG. 4A Example interictal spikes are shown in untreated mice.
  • FIG. 4B Counting interictal spikes in untreated and treated (3e10 gc each DLX2.0 N+C SCN1A AAV vector delivered BL-ICV at P0-P3) mice reveals a significantly decreased frequency of interictal spikes with treatment.
  • n 10 animals per condition, each circle represents one animal, bars and error bars represent means and standard error of the means.
  • FIG.4C-4D Spontaneous MC event prevention in DS model mice.
  • FIG.4C Example MC event observed in untreated Scn1afl/+; Meox2-Cre DS model mice, which have sharp spikes followed closely by EMG signals.
  • FIG. 4D We categorized mice as having MC events or not having MC events during the recording period, which revealed a highly significant prevention of MCs in the treated animals.
  • *** p 7.1e-4 by Fisher’s exact test.
  • FIG.4E-4F Spontaneous GTCs in DS model mice.
  • FIG.4E Example spontaneous GTC seizure observed in an untreated Scn1afl/+; Meox2-Cre DS model mice.
  • Figures 5A-5D Recovery of severe epileptic phenotypes in mice lacking SCN1A in telencephalic GABAergic interneurons using DLX2.0-SCN1A AAVs.
  • FIG. 5A Genetic cross resulting in 100% Scn1a+/fl; Dlx5/6-Cre animals.
  • FIG.5C-5D Protection from seizures in treated Scn1a+/fl; Dlx5/6-Cre animals during thermal challenge assay.
  • FIG. 6A-6F Nonselective delivery of SCN1A to neurons using hSyn1 promoter.
  • FIG.6A Recombinant AAV2/PHP.eB vectors for delivery of hSyn1-split-intein- SCN1A.
  • FIG. 6B Efficient SCN1A reconstitution in mouse brain with hSyn1-split-intein- SCN1A vectors.
  • FIG. 6D Representative stitched fluorescence image of biodistribution of biodistribution of HA and FLAG epitopes in neurons after co-injection with N- and C-terminal vectors. Expression is pseudo-colored black. Expression shown at P84.
  • FIG. 6E Representative stitched fluorescence image of HA and FLAG epitopes and NeuN+ neurons throughout the layers of VISp. Expression shown at P84.
  • FIG. 7A-7F Early pre-weaning toxicity and weak protection from epileptic symptoms with nonselective SCN1A vectors.
  • FIG. 7A Experimental timeline to rescue of epileptic symptoms in DS model mice with nonselective hSyn1 promoter-driven SCN1A vectors.
  • FIG. 7B-7C Preweaning mortality in DS model mice after treatment with nonselective SCN1A AAVs.
  • FIG. 7B, 7D-7F represents the same sets of untreated animals as that shown in Fig. 3B-3E.
  • FIG.7C From analysis of recovered genotypes at P21 we inferred that both DS and littermate control animals were similarly affected by nonselective SCN1A AAV lethality. FD: found dead.
  • FIG. 7D-7F Protection from heat-induced seizures in DS model mice after treatment with high-dose nonselective SCN1A AAVs.
  • FIG. 8A-8E Isoform usage and allele prevalence of human SCN1A.
  • FIG. 8A-8B SCN1A isoform usage across cortical cell type subclasses in mice (8A) and humans (8B).
  • Mouse VISp cortical cell type-specific RNA-seq profiles are from Tasic, et al., Nature 563, 72 (2016) and human middle temporal gyrus (MTG) cortical cell type-specific profiles are from Hodge, et al., Nature 573, 61–68 (2019).
  • Genome-aligned reads are aggregated according cell type subclasses, and visualized as pileups on UCSC genome browser alongside the positions of the exon whose splice donor usage determines whether the 2009-, 1998-, or 1981-amino acid isoform of SCN1A is expressed.
  • Full vertical scale represents 0.65 (mouse) or 0.4 (human) read counts per million.
  • FIG.8C Alignment of mammalian SCN1A protein sequences. The alanine residue at position 1056 in the NCBI RefSeq human SCN1A sequence is orthologous to a conserved threonine residue in most other mammalian species. Additionally, Origene commercial clones of human SCN1A contain threonine residue at this position, but agree at all other positions.
  • DNA sequences represent unique (non-PCR duplicate) reads from assay for transposase- accessible chromatin with sequencing (ATAC-seq) from three independent human patient brain samples (H17.26.001, H17.26.003, H18.03.001).
  • FIG.8E Population allele frequencies in human SCN1A via gnomAD database.
  • gnomAD v3 and v2 populations represent partially overlapping healthy patient populations subject to genome and/or exome sequencing.
  • the threonine-encoding allele is represented by a T on the + strand (corresponding to A on the - strand) in 73-74% of the population.
  • Figures 9A-9C Cell class specificity observed with an independent marker of telencephalic GABAergic interneurons.
  • We injected these mice at P2 BL-ICV with 3e10 gc each DLX2.0-SCN1A AAV vector produced at PackGene and analyzed expression at P30-P35 with both sagittal and coronal sections (n 4 mice analyzed).
  • SS somatosensory cortex
  • MC motor cortex
  • FIG. 9A-9C Cell class specificity observed with an independent marker of telencephalic GABAergic interneurons.
  • FIG. 10A-10E Biodistribution and rescue of epileptic symptoms from independently produced batches of DLX2.0-split intein SCN1A vectors. Specific expression of HA-tagged N-terminal and FLAG-tagged C-terminal SCN1A half-channels in Gad67+ neurons with independent packaging of DLX2.0-SCN1A AAVs. Animals were dosed at low dose (1e10 gc each vector) or high dose (3e10 gc each vector) of DLX2.0-SCN1A AAVs by BL-ICV at P0-P3.
  • FIG.10A Telencephalic GABAergic interneuron specificity is maintained while completeness increases with greater dose of AAV.
  • FIG. 11A Testing DLX2.0-split-intein-SCN1A in an independent mouse model of DS.
  • FIG. 11A Testing DLX2.0-split-intein-SCN1A in an independent mouse model of DS.
  • Example #1 untreated Scn1a+/R613X mouse displays several non-uniformly distributed GTC seizures over 228 hours of recording (one GTC seizure shown), as well as interictal spikes marked by red dots (zoom in on one example spike).
  • Example #2 N+C DLX2.0-SCN1A-injected Scn1a+/R613X mouse displays no GTCs and few spikes.
  • Example #3 N+C DLX2.0-SCN1A-injected Scn1a+/R613X mouse shows no GTCs but frequent spikes with aberrant spike morphology, likely an outlier.
  • FIG. 11D Protection from GTCs with treatment in DS model mice.
  • Intein refers to a polypeptide sequence capable of catalyzing a protein splicing reaction that excises its (the intein) sequence from their host protein and joins flanking sequences (N- and C-exteins) with a peptide bond. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called “protein splicing,” by analogy to the splicing of RNA introns from pre-mRNA (Perler F et al, Nucl Acids Res.22: 1125-1127 (1994)).
  • the segments are called “intein” for internal protein sequence, and “extein” for external protein sequence, with upstream exteins termed “N- exteins” and downstream exteins called “C-exteins.”
  • the products of the protein splicing process are two stable proteins: the mature protein and the intein.
  • Inteins are typically 150-550 amino acids in size and may also contain a homing endonuclease domain.
  • a list of known inteins, and exemplary mutually orthogonal split inteins, are shown at www.inteins.com, described by Pinto et al. in Nature Communications 2020, 11:1529, and provided in for example US2023/0116688, which is incorporated by reference herein.
  • inteins share a low degree of sequence similarity, with conserved residues only at the N- and C-termini. Most inteins begin with Ser or Cys and end in His-Asn or in His-Gln. In various embodiments, the first amino acid of the C-extein is an invariant Ser, Thr, or Cys, but the residue preceding the intein at the N-extein is not conserved.
  • split intein refers to any intein in which the N- terminal and C-terminal amino acid sequences are not directly linked via a peptide bond, such that the N-terminal and C-terminal sequences become separate fragments that can non- covalently re-associate, or reconstitute, into an intein that is functional for trans-splicing reactions.
  • a split intein involves two complementary half inteins, termed the N-intein and C-intein, that associate selectively and extremely tightly to form an active intein enzyme (Shah N.H., et al, J. Amer. Chem.
  • split inteins self-associate and catalyze protein-splicing activity in trans.
  • Split inteins have been identified in diverse cyanobacteria and archaea (Caspi et al, Mol Microbiol.50: 1569-1577 (2003); Choi J. et al, J Mol Biol.556: 1093- 1106 (2006.); Dassa B. et al, Biochemistry.46:322-330 (2007.); Liu X.
  • DNA helicases gp41-l, gp41-8
  • Inosine-5 '-monophosphate dehydrogenase IMPDH-1
  • Ribonucleotide reductase catalytic subunits NrdA-2 and NrdJ-1
  • split intein N-fragment refers to any intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions, that is, that is capable of associating with a functional split intein C-fragment to form a complete intein that is capable of excising itself from the host protein, catalyzing the ligation of the extein or flanking sequences with a peptide bond, or that upon association with a split intein C-fragment catalyzes the “N-terminal cleavage”, that is, the nucleophilic attack of the peptide bond between the extein and the N- terminus of the split intein N-fragment resulting in the breaking of said peptide bond.
  • An lntN thus also comprises a sequence that is spliced out when trans-splicing occurs.
  • An lntN can comprise a sequence that is a modification of the N-terminal portion of a naturally occurring intein sequence. For example, it can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the lntN non-functional in trans-splicing.
  • the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the lntN.
  • N-intein is fused to the N-terminal fragment of a protein to be reconstituted, wherein the N- intein is at the C-teminus relative to the N-fragment of the protein to be reconstituted.
  • split intein C-fragment refers to any intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions, that is, that upon association is capable of associating with a functional split intein N-fragment to form a complete intein that is capable of excising itself from the host protein, catalyzing the ligation of the extein or flanking sequences with a peptide bond, or that upon association with a split N-intein cataly
  • An lntC thus also comprises a sequence that is spliced out when trans-splicing occurs.
  • An lntC can comprise a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, it can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the lntC non-functional in trans-splicing.
  • the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the lntC.
  • the C-intein is fused to the C-terminal fragment of a protein to be reconstituted, wherein the C- intein is at the N-terminus relative to the C-terminal fragment of the protein to be reconstituted.
  • “Degrons,” “degradation signal,” or “destabilizing domain” refers to a naturally-occurring or artificially-constructed polypeptide sequence which when recombinantly fused to another polypeptide it accelerates its protein degradation via the proteosomal degradation pathway, or any other cellular degradation mechanism.
  • Enhancer or “enhancer element” refers to a cis-acting sequence that increases the level of transcription associated with a promoter and can function in either orientation relative to the promoter and the coding sequence that is to be transcribed and can be located upstream or downstream relative to the promoter or the coding sequence to be transcribed.
  • an “enhancer” is an DNA regulatory element that confer cell type specificity of gene expression.
  • a targeted central nervous system cell type enhancer is an enhancer that is uniquely or predominantly utilized by the targeted central nervous system cell type; and a targeted central nervous system cell type enhancer enhances expression of a gene in the targeted central nervous system cell type, but does not substantially direct expression of genes in other non-targeted cell types, thus having neural specific transcriptional activity.
  • enhancers especially interneuron-specific enhancers, are provided in US2021/0348195 and US2018/0078658, which are incorporated by reference.
  • Neurons found in the mammalian (e.g., human) nervous system can be divided into three classes based on their roles: sensory neurons, motor neurons, and interneurons.
  • Ion transporters are transmembrane proteins that mediate transport of ions across cell membranes.
  • ion transporters include voltage gated sodium channels, potassium channels, and calcium channels.
  • Mammalian voltage-gated sodium (Na v ) channels are composed of a highly glycosylated ⁇ 260 kDa ⁇ subunit, the pore forming protein, linked via disulfide bonds to ⁇ 2/ ⁇ 4 subunits and non-covalently with ⁇ 1/ ⁇ 3 subunits.
  • Nav1 ⁇ subunit genes SCN1A–SCN9A
  • the Nav channel ⁇ subunit is a complex of transmembrane helices surrounding a central ion-conducting pore, usually capable of producing functional channels in a heterologous expression system.
  • the SCN1A gene codes for the alpha subunit of Nav1.1 channel.
  • the Nav1.1 channel is mainly responsible for the generation and propagation of neuronal action potentials. Different mutations in this gene are associated with epilepsy and febrile seizures.
  • SCN1A is part of a cluster of voltage-gated sodium channel genes that is home to SCN2A, SCN3A, SCN7A, as well as SCN9A, which encode Nav1.2, Nav1.3, Nax, and Nav1.7, respectively.
  • Coding sequences encoding molecules e.g., RNA, proteins
  • Coding sequences can be readily obtained from publicly available databases and publications. Coding sequences can further include various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the encoded molecule.
  • encode refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.
  • gene may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. The sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.
  • vectors refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct.
  • vectors include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
  • plasmids e.g., DNA plasmids or RNA plasmids
  • transposons e.g., DNA plasmids or RNA plasmids
  • cosmids e.g., bacterial artificial chromosomes
  • viral vectors e.g., viral vectors.
  • Addenovirus vectors refer to those constructs containing adenovirus sequences sufficient to support packaging of an expression construct and to express a coding sequence that has been cloned therein in a sense or antisense orientation.
  • a recombinant Adenovirus vector includes a genetically engineered form of an adenovirus. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut-off.
  • vectors e.g., AAV
  • BBB blood-brain barrier
  • vectors are modified to include capsids that cross the BBB.
  • AAV with viral capsids that cross the blood brain barrier include AAV9, AAVrh.10, AAV1R6, AAV1R7, rAAVrh.8, AAV-BR1, AAV- PHP.S, AAV-PHP.B, and AAV-PPS.
  • the PHP.eB capsid differs from AAV9 such that, using AAV9 as a reference, amino acids starting at residue 586: S-AQ-A (SEQ ID NO:46) are changed to S-DGTLAVPFK-A (SEQ ID NO:47). Additional description regarding capsids that cross the blood brain barrier is provided by Chan et al., Nat. Neurosci. 2017 August: 20(8): 1172-1179. [0079] Various embodiments provide the inclusion of a degradation signal in a system, wherein the degradation signal is a peptide sequence, known as degron, which mediates rapid ubiquitination and subsequent proteasomal degradation of a nearby protein or a protein that the degron is embedded in.
  • the degradation signal is a peptide sequence, known as degron, which mediates rapid ubiquitination and subsequent proteasomal degradation of a nearby protein or a protein that the degron is embedded in.
  • the degradation signal, or degron is included with the N-terminal segment (split) of an intein, i.e., N-intein.
  • the drgron is included with the C-terminal segment (split) of the intein, i.e., C-intein.
  • the degron is included with both the N-intein and the C-intein.
  • the degron is in an individual expression construct or vector, separate from the expression construct(s) or vector(s) that contain C-intein or N-intein.
  • a degron is included with (or attached to) just one of the C-intein or the N-intein, and not with both the C-intein and the N-intein.
  • the degron when a degron is attached to the N-intein, the degron is attached to the C-terminal end of the N-intein, and the configuration from N- to C-end of this half of the split intein fusion is: N- terminal fragment of a sodium channel protein (e.g., N-terminal fragment of a sodium channel alpha subunit) – N-intein – degron.
  • the degron when a degron is included with (or attached to) the C-intein, the degron is inserted into the C-intein and a few residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9.10 residues) before/upstream/at the N-terminus relative to the fragment of the sodium channel protein; and that is, the configuration from N to C is: a first portion of C-intein – degron – a second portion of the C-intein – C-terminal end containing fragment of the sodium channel protein.
  • the first portion of C-intein and the second portion of the C-intein when operably connected, form a C-intein.
  • the degradation signal, or degron is integrated at the 5’ end of the N-intein, so that upon intein-mediated protein trans-splicing and intein excision, the degron would be placed at the N-terminal of the excised intein.
  • the degradation signal, or degron is integrated at the 3’ end of the C-intein, so that upon intein-mediated protein trans-splicing and intein excision, the degron would be placed at the C-terminal of the excised intein.
  • the degradation signal, or degron is integrated at the 3’ end of the N-intein and/or the 5’ end of the C-intein, so that upon intein-mediated protein trans-splicing and intein excision, the degron would be in the middle of the excised intein.
  • the degron encoded in the system is one having no more than 30 amino acid residues.
  • the degron encoded in the system is one being 5- 27 amino-acid residues in length.
  • the degron encoded in the system is one being 5-10 amino-acid residues in length.
  • the degron encoded in the system is one being 11-20 amino-acid residues in length.
  • the degron encoded in the system is one being 21-26 amino-acid residues in length.
  • the system includes polynucleotides encoding a short degron (e.g., no more than 30 amino-acid residues in length) and an intein having fast splicing rates (e.g., half-lives below 5 min) such as consensus fast intein (Cfa); and the system does not include polynucleotides encoding degron that is more than 30 amino-acid residues in length or polynucleotides encoding segments that form an intein with a splicing rate slower than Cfa or half-lives greater than 5 min.
  • a short degron e.g., no more than 30 amino-acid residues in length
  • an intein having fast splicing rates e.g., half-lives below 5 min
  • Cfa consensus fast intein
  • the system is effective for reconstituting a target protein (e.g., sodium channel alpha subunit) at a faster speed or higher efficiency than degron-mediated degradation of intein, N-intein and/or C-intein.
  • a target protein e.g., sodium channel alpha subunit
  • the system having a polynucleotide sequence encoding the degron is effective for reconstituting a target protein (e.g., sodium channel alpha subunit) at an amount or yield that is at least 100%, 95%, 90%, 85%, or 80% compared to that reconstituted in a system without a polynucleotide sequence.
  • the system having a polynucleotide sequence encoding the degron is effective for reducing amount of intein (e.g., free intein after target protein splicing) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • a degron is from the class II trans-activator (CIITA). It some embodiments, the CIITA degron is a 26 amino acid-long peptide, having an amino acid sequence of RPGSTSPFAPSATDLPSMPEPALTSR (SEQ ID NO:91). In some embodiments, the CIITA degron is a variant having at least 95% or 90% sequence identity with SEQ ID NO:91.
  • the CITTA degron is a N-terminal degron. That is, preferably, when the CITTA degron is attached to the N-intein, the CITTA degron is at the C-terminal of the N-intein, and the configuration from N to C is: N-terminal fragment of the sodium channel alpha subunit – N-intein – CITTA degron; and the other half of the split-intein has a configuration from N to C being C-intein – C-terminal fragment of the sodium channel alpha subunit.
  • the CITTA degron is inserted within the C-intein, and the configuration from N to C of this half of the split intein fusion is: a first portion of C-intein – degron – a second portion of the C-intein – C-terminal fragment of the sodium channel alpha subunit; and the other half of the split-intein has a configuration from N to C being N-terminal fragment of the sodium channel alpha subunit – N-intein.
  • a degron is derived from the ornithine decarboxylase 1 (ODC1).
  • the ODC1 degron is a 7 amino acid-long peptide, having an amino acid sequence of MSCAQES (SEQ ID NO:90). In some embodiments, the ODC1 degron is a variant having at least 95% or 90% sequence identity with SEQ ID NO:90. It has been determined that part of some degron sequences do not participate in the binding with ubiquitin ligase; and hence, a shorter fragment of some long degron sequences may be used which involves in binding with ubiquitin ligase to mediate degradation. For example, an active (ligase-binding) fragment of the ODC1 degron consists of an amino acid sequence of MSCAQES.
  • one or more ODC1 degrons are each a C-terminal degron. That is, preferably, the ODC1 degron is attached to as inserted into the C-intein, and the configuration from N to C of this half of the split intein fusion is: a first portion of C-intein – degron – a second portion of the C-intein – C-terminal fragment of the sodium channel alpha subunit; and the other half of the split-intein has a configuration from N to C being N-terminal fragment of the sodium channel alpha subunit – N-intein.
  • a degron is the peptide CL1 or a variant thereof.
  • the CL1 degron is a 16 amino acid-long peptide, having an amino acid sequence of ACKNWFSSLSHFVIHL (SEQ ID NO:92). In some embodiments, the CL1 degron is a variant having at least 95% or 90% sequence identity with SEQ ID NO:92. In some embodiments, the CL1 degron or a variant thereof (“CL degron”) is attached to the N-intein and at the C-terminal of the N-intein, i.e., as a C-terminal tail of N-intein.
  • a configuration from N- to C-terminus is: N-terminal fragment of the sodium channel alpha subunit – N-intein – CL degron; and the other half of the split-intein has a configuration from N to C being C-intein – C-terminal fragment of the sodium channel alpha subunit.
  • Description of the CL degron is further provided by Gilon et al., the EMBO Journal, Vol.17 No.10 pp.2759–2766, 1998.
  • Variants of peptide CL1 include CL2, CL6, CL9, CL10, CL11, CL12, CL15, CL16, and SL17, whose sequences are summarized below, as well as those having at least 95% or 90% sequence identity thereto: CL2 SLISLPLPTRVKFSSLLLIRIMKIITMTFPKKLRS (SEQ ID NO:94) CL6 FYYPIWFARVLLVHYQ (SEQ ID NO:95) CL9 SNPFSSLFGASLLIDSVSLKSNWDTSSSSCLISFFSSVMFSSTTRS (SEQ ID NO:96) CL10 CRQRFSCHLTASYPQSTVTPFLAFLRRDFFFLRHNSSAD (SEQ ID NO:97) CL11 GAPHVVLFDFELRITNPLSHIQSVSLQITLIFCSLPSLILSKFLQV (SEQ ID NO:98) CL12 NTPLFSKSFSTTCGVAKKTLLLAQISSLFFLLLSSNIAV (SEQ ID NO:99) CL15 PTVKNSPK
  • the DEG1 degron is a 9 amino acid-long peptide, having an amino acid sequence of GSLIIFIIL (SEQ ID NO:93). In some embodiments, the DEG1 degron is a variant having at least 95% or 90% sequence identity with SEQ ID NO:93. In some embodiments, the DEG1 degron is a C-terminal degron.
  • the DEG1 degron is inserted in the C-intein, and the configuration from N to C of this half of the split intein fusion is: a first portion of C-intein – degron – a second portion of the C-intein – C-terminal fragment of the sodium channel alpha subunit; and the other half of the split-intein has a configuration from N to C being N-terminal fragment of the sodium channel alpha subunit – N-intein.
  • Various embodiments provide systems to express a coding sequence of a sodium channel alpha subunit (e.g., Na v 1.1 ⁇ subunit, or short as Na v 1.1 unless otherwise noted) for reconstitution of the sodium channel alpha subunit.
  • the system expresses a coding sequence of Nav1.1, which is gene SCN1A.
  • the systems express the coding sequence of a sodium channel alpha subunit selected from Nav1.1 through Nav1.9, and correspondingly encoded by genes SCN1A through SCN11A.
  • the systems express SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, SCN11A, or SCN7A, for reconstitution of Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, or Nax, respectively.
  • a system to express a coding sequence of Nav1.1 alpha subunit wherein the coding sequence of Nav1.1 alpha subunit is or comprises the polynucleotide sequence of SCN1A, and the system includes: a first expression construct including a first portion of the polynucleotide sequence of the SCN1A, and a polynucleotide sequence encoding an N-fragment of a split intein (N-intein) at the 3’ end relative to the first portion of the polynucleotide sequence of the SCN1A; and a second expression construct including a second portion of the polynucleotide sequence of the SCN1A, and a polynucleotide sequence encoding a C-fragment of the split intein (C-intein) at the 5’ end relative to the second portion of the polynucleotide sequence of the SCN1A; wherein protein products of the first and the second portions of the polynucle
  • a composition or a system for reconstitution of Nav1.1 alpha subunit, which comprises: a. a first polynucleotide encoding a polypeptide comprising an N-fragment of a split intein, wherein the N-fragment of the split intein is directly linked via a peptide bond, optionally through a peptide linker, to the N-terminal fragment of Nav1.1 alpha subunit; b.
  • a second polynucleotide encoding a polypeptide comprising a C-fragment of the split intein, wherein the C-fragment of the split intein is directly linked via a peptide bond, optionally through a peptide linker, to the C-terminal fragment of the Nav1.1 alpha subunit; and c.
  • a third polynucleotide encoding a degron; wherein the polynucleotides of the composition may be packed together in a single formulation or separately in different formulations, wherein the first and the second polynucleotides encode the N- and the C-terminal fragments of the Nav1.1 alpha subunit, respectively, so that when both fragments are spliced together, the N-terminal fragment is linked to the C-terminal fragment, generating whole Nav1.1 alpha subunit.
  • the composition is further characterized in that: the split intein N-fragment is further directly linked via a peptide bond to a degron, wherein the degron is linked to the intein N-fragment via the C-terminus of the intein, with or without a linker between the intein N-fragment and the degron, and wherein the N-terminus of the Split intein N-fragment is directly linked via a peptide bond to the N-terminal fragment of the Nav1.1 alpha subunit; and/or the split intein C-fragment is further directly linked via a peptide bond to a degron, wherein the degron is linked to the intein C-fragment via the N-terminus of the intein, with or without a linker between the intein C-fragment and the degron, and wherein the C-terminus of the Split intein C-fragment is directly linked via a peptide bond to the C-terminal fragment of the a degro
  • a system to express a coding sequence of Nav1.1 for its reconstitution includes a first expression construct and a second expression construct, wherein the first expression constructs includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the N-intein – a polynucleotide sequence encoding the degron; and wherein the second expression constructs includes from 5’ to 3’ end: a polynucleotide sequence encoding a C-fragment of the split intein (C-intein) – a second portion of the polynucleotide sequence of the SCN1A.
  • the first expression constructs includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the N-intein – a polynucleotide sequence encoding the degron
  • the polynucleotide sequence encoding the degron is one that encodes RPGSTSPFAPSATDLPSMPEPALTSR (SEQ ID NO:91) or ACKNWFSSLSHFVIHL (SEQ ID NO:92) or a variant having a sequence identity of at least 90% or 95% thereto.
  • a system to express a coding sequence of Nav1.1 for its reconstitution includes a first expression construct and a second expression construct, wherein the first expression constructs includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the degron – a polynucleotide sequence encoding the N-intein; and wherein the second expression constructs includes from 5’ to 3’ end: a polynucleotide sequence encoding a C-fragment of the split intein (C-intein) – a second portion of the polynucleotide sequence of the SCN1A.
  • the first expression constructs includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the degron – a polynucleotide sequence encoding the N-intein
  • a system to express a coding sequence of Nav1.1 for its reconstitution includes a first expression construct and a second expression construct, wherein the first expression construct includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the N-intein; and the second expression construct includes from 5’ to 3’: a polynucleotide sequence encoding the degron – a polynucleotide sequence encoding the C-intein – a second portion of the polynucleotide sequence of the SCN1A.
  • the polynucleotide sequence encoding the degron is one encoding MSCAQESITSLYKKAGSENLYFQ (SEQ ID NO:88), FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO:89), MSCAQES (SEQ ID NO:90), or GSLIIFIIL (SEQ ID NO:93), or a sequence having at least 90% or 95% sequence identity thereto.
  • a system to express a coding sequence of Nav1.1 for its reconstitution includes a first expression construct and a second expression construct, wherein the first expression construct includes from 5’ to 3’: a first portion of the polynucleotide sequence of the SCN1A – a polynucleotide sequence encoding the N-intein; and wherein the second expression construct includes from 5’ to 3’: a polynucleotide sequence encoding the C- intein – a polynucleotide sequence encoding the degron – a second portion of the polynucleotide sequence of the SCN1A.
  • the degron has an amino acid sequence selected from the group consisting of: MSCAQESITSLYKKAGSENLYFQ (SEQ ID NO:88), FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO:89), MSCAQES (SEQ ID NO:90), RPGSTSPFAPSATDLPSMPEPALTSR (SEQ ID NO:91), ACKNWFSSLSHFVIHL (SEQ ID NO:92), and GSLIIFIIL (SEQ ID NO:93), and variants having at least 90% or 90% sequence identity to any of the above.
  • Alternative sites are provided for splitting human SCN1A to make AAV-sized halves.
  • the protein is split at (right before, i.e., at the N-terminus end before) breakpoint Cys1050, according to amino acid positions in sodium channel protein type 1 subunit alpha isoform 2 of NCBI reference no. NP_001340878.1.
  • an endogenous cysteine residue is required to make half joining scarless and reconstitute a full- length unmutated protein.
  • Alternative split intein breakpoints are at either Cys957 or Cys948, according to amino acid positions in sodium channel protein type 1 subunit alpha isoform 2 of NCBI reference no. NP_001340878.1.
  • breakpoints would permit a better AAV size and packaging efficiency of the N-terminal half for better expression than that seen with hSCN1A- CO-Nterm1049 when using the Cys1050 breakpoint.
  • they would place the intein junctions in the extracellular/lumenal space.
  • the N-terminal junction sequence at the front of an intein is termed as the ⁇ 1 position; and the +1 position after the intein sequence usually has a Cys, Ser, or Thr residue.
  • the first portion of the polynucleotide sequence of the SCN1A encodes residues 1-1049 of the Nav1.1
  • the second portion of the polynucleotide sequence of the SCN1A encodes residues 1050- 1998 of the Nav1.1, wherein the amino acid position is based on numberings in NP_001340878.1
  • the first portion of the polynucleotide sequence encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 1-1049 of the Nav1.1 having an NCBI reference no.
  • the second of portion of the polynucleotide sequence encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 1050-1998 of the Nav1.1 having an NCBI reference no. NP_001340878.1.
  • the first portion of the polynucleotide sequence of the SCN1A encodes residues 1-956 of the Nav1.1
  • the second portion of the polynucleotide sequence of the SCN1A encodes residues 957- 1998, wherein the amino acid position is based on numberings in NP_001340878.1.
  • the first portion of the polynucleotide sequence encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 1-956 of the Nav1.1 having an NCBI reference no. NP_001340878.1
  • the second of portion of the polynucleotide sequence encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 957-1998 of the Nav1.1 having an NCBI reference no. NP_001340878.1.
  • the first portion of the polynucleotide sequence of the SCN1A encodes residues 1-947 of the Nav1.1, and the second portion of the polynucleotide sequence of the SCN1A encodes residues 948- 1998, wherein the amino acid position is based on numberings in NP_001340878.1.
  • the first portion of the polynucleotide sequence encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 1-947 of the Nav1.1 having an NCBI reference no.
  • NP_001340878.1 encodes a sodium channel alpha subunit N-terminus fragment when sequence aligned being corresponding to residues 947-1998 of the Nav1.1 having an NCBI reference no. NP_001340878.1.
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm1049 (SEQ ID NO: 59), and the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm949 (SEQ ID NO: 60).
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm956 (SEQ ID NO: 61), and the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm1042 (SEQ ID NO: 62).
  • the first portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Nterm947 (SEQ ID NO: 63)
  • the second portion of the polynucleotide sequence of the SCN1A comprises a polynucleotide sequence of hSCN1A-CO-Cterm1051 (SEQ ID NO: 64).
  • the intein includes a Cfa intein, an Ssp intein, a gp41-1 intein, IMPDH-1 intein, Nrdj-1 intein, gp41-8 intein, or an Npu intein.
  • the split intein comprises consensus fast intein (Cfa),. We conceive that with the Cfa, the split intein trans-splicing reaction will occur first, before the binding of ubiquitin ligase to degron and subsequent degradation.
  • the polynucleotide sequence encoding the N- intein comprises a polynucleotide sequence of Cfa-N (SEQ ID NO:57), and the polynucleotide sequence encoding the C-intein comprises a polynucleotide sequence of Cfa-C (SEQ ID NO: 58).
  • the intein is functionally similar to a Cfa intein.
  • functionally similar to a Cfa intein means that the expression construct includes a variant of a Cfa intein, yet still results in construction of a functional protein (e.g., voltage-gated sodium channel).
  • a mature protein Nav1.1 is expressed by splitting the coding sequence into three fragments and putting the N-terminal portion of the coding sequence with a first N-intein into a first artificial expression construct, putting the middle portion of the coding sequence with a first C-intein and second N-intein into a second artificial expression construct, and putting the C-terminal portion of the coding sequence with a second C-intein into a second artificial expression construct, wherein the first N-intein and first C-intein specifically splice together to form an intein, and wherein the second N-intein and second C- intein specifically splice together to form an intein.
  • At least one, or two or all three fragments of the coding sequence includes a degron.
  • the first intein and the second intein are different, so that the first N-intein and the second C-intein do not splice together, and the second N-intein and the first C-intein do not splice together. That is, preferably, first N-intein and first C-intein, and second N-intein and second C-intein, are two mutually orthogonal split inteins. Exemplary mutually orthogonal split inteins are described at least by Pinto et al. in Nature Communications (2020)11:1529.
  • a method of using this system includes administering the first, second, and third artificial expression construct to a cell.
  • mature proteins can be formed from several fragments using the appropriate number of inteins.
  • the first expression construct and the second expression construct independently further comprise a promoter sequence, selected from a minBglobin promoter, an hSyn1 promoter, or a CMV promoter; optionally the hSyn1 promoter comprising a shortened hSyn1 promoter having a polynucleotide sequence of SEQ ID NO:54.
  • the first expression construct, the second expression construct, or both independently further comprise an enhancer sequence, configured for targeted expression of the first, the second, or both portions, respectively, of the polynucleotide sequence of the sodium channel alpha subunit within a targeted central nervous system cell type.
  • the enhancer sequence comprises a polynucleotide sequence of DLX2.0 (SEQ ID NO:2).
  • the enhancer sequence has a concatemerized core of a I56i enhancer optionally as set forth in SEQ ID NO:1.
  • the targeted central nervous system cell type comprises a GABAergic neuron, preferably a GABAergic interneuron, or more preferably a telencephalic GABAergic interneuron.
  • the enhancer sequence is a 527 bp enhancer sequence (referred to as mI56i or mDIx) from the intergenic interval between the distal-less homeobox 5 and 6 genes (DIx5/6), which are naturally expressed by forebrain GABAergic interneurons during embryonic development. Further description of enhance sequences, such as those for selectively modulating gene expression in interneurons, is provided in US20210348195, which is incorporated by reference.
  • the enhancer sequence comprises a polynucleotide sequence of eHGT_078h (SEQ ID NO:55).
  • the targeted central nervous system cell type comprises a forebrain glutamatergic neuron.
  • the first expression construct, the second expression construct, or both independently further comprise a miRNA binding site sequence, configured for targeted expression of the first, the second, or both portions, respectively, of the polynucleotide sequence of the sodium channel alpha subunit within a selected central nervous system cell type.
  • the miRNA binding site sequence comprises a polynucleotide sequence of 4x2C miRNA binding site (SEQ ID NO:56), and the selected central nervous system cell type comprises a pan-GABAergic neuron.
  • an enhancer is used to drive gene expression in a targeted central nervous system cell population.
  • the artificial expression constructs utilize the following enhancers to drive gene expression within targeted central nervous system cell populations as follows (enhancer / targeted cell population): DLX2.0 / forebrain GABAergic; hSyn1 with 4x2C or 8x2C miR binding site / pan-GABAergic neurons; eHGT_078h / forebrain glutamatergic neurons.
  • the artificial expression construct can include a shortened promoter or a minimal promoter.
  • the shortened promoter includes the hSyn1 promoter(shortened).
  • the minimal promoter includes minBglobin.
  • inventions provide artificial expression construct pairs including the features of vectors described herein including vectors: CN3252 and CN3254, CN3683 and CN3684, CN3251 and CN3253, CN3677 and CN3678, CN4541 and CN4542, CN4217 and CN4218, or CN4642 and CN4643, as described in Tables below.
  • Various embodiments further provide an artificial expression construct containing a first expression construct as disclosed herein.
  • Further embodiments also provide an artificial expression construct containing a second expression construct as disclosed herein.
  • the artificial expression construct is within an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • the artificial expression construct can also include other regulatory elements if necessary or beneficial.
  • the artificial expression constructs are expressed in all neurons.
  • the artificial expression constructs include an hSyn1 promoter and are expressed in neurons.
  • the artificial expression constructs are expressed in all cell lines.
  • the artificial expression constructs include a CMV promoter and are expressed in cell lines.
  • Various embodiments provide an administrable composition, which includes any one or more systems disclosed herein to express coding sequence of and reconstitute Nav1.1. Additional embodiments provide an administrable composition, which includes either one or both of an artificial expression construct containing the first expression construct, and an artificial expression construct containing the second expression construct.
  • a transgenic cell is also provided, comprising any one or more systems disclosed herein. In some aspects, the transgenic cell is a GABAergic neuron or a glutamatergic neuron or a cell line of GABAergic neuron or glutamatergic neuron.
  • Additional embodiments provide a method of rescuing voltage-gated sodium channel function in a population of cells, and the method includes co-administering a therapeutically effective amount of a first expression construct and a therapeutically effective amount of a second expression construct, as disclosed herein, to a sample or subject comprising the population of cells, and inducing expression of the first expression construct and the second expression construct to reconstitute Nav1.1, thereby rescuing voltage-gated sodium channel function in the population of cells.
  • the method is for rescuing voltage-gated sodium channel function in a targeted population of cells.
  • the methods involve a targeted central nervous system cell type enhancer, which is uniquely or predominantly utilized by the targeted central nervous system cell type.
  • a targeted central nervous system cell type enhancer enhances expression of a gene in the targeted central nervous system.
  • a targeted central nervous system cell type enhancer is also a targeted central nervous system type enhancer that enhances expression of a gene in the targeted central nervous system and does not substantially direct expression of genes in other non-targeted cell types, thus having cell type specific transcriptional activity.
  • the subject has an SCN1A-related seizure disorder comprising Dravet syndrome, myoclonic seizures, myoclonic astatic epilepsy (MAE), intractable childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epilepsy and febrile seizures plus (GEFS+), migrating partial seizures of infancy, Lennox-Gastaut syndrome, or West syndrome.
  • the subject is a pediatric patient having Dravet syndrome.
  • the subject is a pediatric human.
  • the subject is an infant (1 year old or younger).
  • the subject is a young child, e.g., between 1 and 10 years old. In some embodiments, the is a teenager. In various embodiments, the human subject is age 1 day through 5 months, 6 months through 4 years, 5 years through 11 years, or 12 years through 17 years.
  • artificial expression constructs can deliver SCN1A as several fragments of SCN1A delivered by several artificial expression constructs.
  • SCN1A can be delivered in a first artificial expression construct including a first portion of the SCN1A coding sequence and second artificial expression construct including a second portion of the SCN1A coding sequence.
  • the first portion of the SCN1A coding sequence is the N-terminal portion of the coding sequence and the second portion of the SCN1A coding sequence is the C-terminal portion of the coding sequence.
  • the sodium channel alpha subunit coding sequence can be split into an N- terminal portion and C-terminal portion at any point, or preferably at a breakpoint wherein the first amino acid residue encoded downstream of the breakpoint is Cys, Ser, or Thr, such that upon intein fusion, a functional sodium channel alpha subunit molecule is expressed.
  • an N-terminal portion of the SCN1A coding sequence includes hSCN1A-CO-Nterm1049 (SEQ ID NO:59), hSCN1A-CO-Nterm956 (SEQ ID NO:61), or hSCN1A-CO-Nterm947 (SEQ ID NO:63).
  • a C-terminal portion of the SCN1A coding sequence includes hSCN1A- CO-Cterm949 (SEQ ID NO:60), hSCN1A- CO-Cterm1042 (SEQ ID NO:62), or hSCN1A-CO- Cterm1051 (SEQ ID NO:64).
  • Exemplary reporter genes/proteins include those expressed by Addgene ID#s 83894 (pAAV-hDlx-Flex-dTomato-Fishell_7), 83895 (pAAV-hDlx-Flex-GFP-Fishell_6), 83896 (pAAV-hDlx-GiDREADD-dTomato-Fishell-5), 83898 (pAAV-mDlx-ChR2- mCherry-Fishell-3), 83899 (pAAV-mDlx-GCaMP6f-Fishell-2), 83900 (pAAV-mDlx-GFP- Fishell-1), and 89897 (pcDNA3- FLAG-mTET2 (N500)).
  • Exemplary reporter genes particularly can include those which encode an expressible fluorescent protein, or expressible biotin; blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T- sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g.
  • blue fluorescent proteins e.g. eBFP, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T- sapphire
  • cyan fluorescent proteins e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise
  • green fluorescent proteins e.g.
  • artificial expression constructs can include DNA and RNA editing tools such CRISPR/Cas (e.g., guide RNA and a nuclease, such as Cas, Cas9 or cpf1).
  • Functional molecules can also include engineered Cpf1s such as those described in US 2018/0030425, US 2016/0208243, WO/2017/184768 and Zetsche et al. (2015) Cell 163: 759-771; single gRNA (see e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al.
  • artificial expression constructs can include tag cassettes.
  • a tag cassette includes His tag (HHHHHH; SEQ ID NO:34), Flag tag (DYKDDDDK; SEQ ID NO:35), Xpress tag (DLYDDDDK; SEQ ID NO:36), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO:37), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO:38), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO:39), Myc tag (EQKLISEEDL; SEQ ID NO:40), Strep tag (which refers the original STREP ® tag (WRHPQFGG; SEQ ID NO:41), STREP ® tag II (WSHPQFEK SEQ ID NO:42 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO:43), Softag 3 (TQDPSRVG; SEQ ID NO:
  • a tag cassette includes a fusion of tag cassettes such as 3XFLAG.
  • 3XFLAG includes the sequence set forth in SEQ ID NO:15.
  • the artificial expression constructs include an internal ribosome entry site (IRES) sequence. See for example, figure 1B. IRES allow ribosomes to initiate translation at a second internal site on a mRNA molecule, leading to production of two proteins from one mRNA.
  • IRES includes IRES2.
  • IRES2 allows for a second protein open reading frame (ORF) to be translated from the same transcript.
  • Coding sequences encoding molecules e.g., RNA, proteins
  • Coding sequences can be obtained from publicly available databases and publications. Coding sequences can further include various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the encoded molecule.
  • the term “encode” or “encoding” refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.
  • the term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and/or post-regulatory elements, such as termination regions.
  • the term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites.
  • the sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.
  • Promoters can include general promoters, tissue-specific promoters, cell- specific promoters, and/or promoters specific for the cytoplasm.
  • Promoters may include strong promoters, weak promoters, constitutive expression promoters, and/or inducible promoters. Inducible promoters direct expression in response to certain conditions, signals or cellular events.
  • the promoter may be an inducible promoter that requires a particular ligand, small molecule, transcription factor or hormone protein in order to effect transcription from the promoter.
  • promoters include minBglobin (also referred to as minBGprom), CMV promoter, hSyn1 promoter, hSyn1 promoter (shortened), minCMV, minCMV* (minCMV* is minCMV with a SacI restriction site removed), minRho, minRho* (minRho* is minRho with a SacI restriction site removed), SV40 immediately early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous Sarcoma Virus (RSV) long-terminal repeat (LTR) promoter.
  • Minimal promoters have no activity to drive gene expression on their own but can be activated to drive gene expression when linked to a proximal enhancer element.
  • expression constructs are provided within vectors.
  • the term vector refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell or may include sequences that permit integration into host cell DNA.
  • Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
  • Adeno-Associated Virus is a parvovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to 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, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter.
  • the AAV DNA is 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three AAV viral promoters have been identified and named p5, p19, and p40, according to their map position.
  • AAVs stand out for use within the current disclosure because of their superb safety profile and because their capsids and genomes can be tailored to allow expression in targeted cell populations.
  • scAAV refers to a self-complementary AAV.
  • pAAV refers to a plasmid adeno-associated virus.
  • rAAV refers to a recombinant adeno-associated virus.
  • pSMART-HCKan is a high copy number vector with a kanamycin resistance marker for efficient blunt cloning of unstable sequences.
  • Other viral vectors may also be employed.
  • vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells [0137] Elements directing the efficient termination and polyadenylation of a heterologous nucleic acid transcript can increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.
  • vectors include a polyadenylation signal 3' of a polynucleotide encoding a molecule (e.g., protein) to be expressed.
  • poly(A) site or "poly(A) sequence” denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
  • Particular embodiments may utilize BGHpA, hGHpA, SV40pA, or shortPolyA.
  • a preferred embodiment of an expression construct includes a terminator element. These elements can serve to enhance transcript levels and to minimize read through from the construct into other plasmid sequences. [0138]
  • Particular embodiments of vectors include:
  • Subcomponent sequences within the larger vector sequences can be readily identified by one of ordinary skill in the art and based on the contents of the current disclosure. Nucleotides between identifiable and enumerated subcomponents reflect restriction enzyme recognition sites used in assembly (cloning) of the constructs, and in some cases, additional nucleotides do not convey any identifiable function. These segments of complete vector sequences can be adjusted based on use of different cloning strategies and/or vectors. In general, short 6-nucleotide palindromic sequences reflect vector construction artifacts that are not important to vector function. [0140] In particular embodiments vectors (e.g., AAV) with capsids that cross the blood-brain barrier (BBB) are selected.
  • AAV blood-brain barrier
  • vectors are modified to include capsids that cross the BBB.
  • AAV with viral capsids that 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), AAV- PHP.B (Deverman et al., Nat Biotechnol.2016; 34(2): 204), AAV-PPS (Chen et al., Nat Med.
  • the PHP.eB capsid differs from AAV9 such that, using AAV9 as a reference, amino acids starting at residue 586: S-AQ-A (SEQ ID NO: 46) are changed to S-DGTLAVPFK-A (SEQ ID NO: 47).
  • PHP.eB refers to SEQ ID NO: 30. Further description of capsids that cross the BBB is provided in US20210348195, which is incorporated by reference. [0141] AAV9 is a naturally occurring AAV serotype that, unlike many other naturally occurring serotypes, can cross the BBB following intravenous injection.
  • AAVrh.10 was originally isolated from rhesus macaques and shows low seropositivity in humans when compared with other common serotypes used for gene delivery applications (Selot et al., Front Pharmacol.
  • AAV1R6 and AAV1R7 two variants isolated from a library of chimeric AAV vectors (AAV1 capsid domains swapped into AAVrh.10), retain the ability to cross the BBB and transduce the CNS while showing significantly reduced hepatic and vascular endothelial transduction.
  • rAAVrh.8 also isolated from rhesus macaques, shows a global transduction of glial and neuronal cell types in regions of clinical importance following peripheral administration and also displays reduced peripheral tissue tropism compared to other vectors.
  • AAV-BR1 is an AAV2 variant displaying the NRGTEWD (SEQ ID NO:48) epitope that was isolated during in vivo screening of a random AAV display peptide library. It shows high specificity accompanied by high transgene expression in the brain with minimal off-target affinity (including for the liver) (Körbelin et al., EMBO Mol Med.2016; 8(6): 609).
  • AAV-PHP.S (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence QAVRTSL (SEQ ID NO:49), transduces neurons in the enteric nervous system, and strongly transduces peripheral sensory afferents entering the spinal cord and brain stem.
  • AAV-PHP.B (Addgene, Watertown, MA) is a variant of AAV9 generated with the CREATE method that encodes the 7-mer sequence TLAVPFK (SEQ ID NO:50). It transfers genes throughout the CNS with higher efficiency than AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.
  • AAV-PPS an AAV2 variant crated by insertion of the DSPAHPS (SEQ ID NO:51) epitope into the capsid of AAV2, shows a dramatically improved brain tropism relative to AAV2.
  • DSPAHPS SEQ ID NO:51
  • a capsid that results in transduction of targeted cell types in a primate following administration e.g., i.v. administration
  • a capsid that results in widespread transduction of tissue and cell types impacted by the loss of Scn1a following administration is chosen.
  • targeted cell types are neurons.
  • neurons include GABAergic neurons or glutamatergic neurons.
  • GABAergic neurons include pan- GABAergic neurons, forebrain GABAergic neurons, hippocampal GABAergic neurons, or cortical GABAergic neurons.
  • glutamatergic neurons include forebrain glutamatergic neurons.
  • Physiologically active components within compositions described herein can be prepared in neutral forms, as freebases, or as pharmacologically acceptable salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. 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 such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • Carriers of physiologically active components can include solvents, dispersion media, vehicles, coatings, diluents, isotonic and absorption delaying agents, buffers, solutions, suspensions, colloids, and the like.
  • the use of such carriers for physiologically active components is well known in the art. Except insofar as any conventional media or agent is incompatible with the physiologically active components, it can be used with compositions as described herein.
  • pharmaceutically-acceptable carriers refer to carriers that do not produce an allergic or similar untoward reaction when administered to a human, and in particular embodiments, when administered intravenously.
  • compositions can be formulated for intravenous, intraparenchymal, intraocular, intravitreal, parenteral, subcutaneous, intracerebro-ventricular, intramuscular, intrathecal, intraspinal, intraperitoneal, oral or nasal inhalation, or by direct injection in or application to one or more cells, tissues, or organs.
  • Compositions may include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and/or nanoparticles.
  • the formation and use of liposomes is generally known to those of skill in the art. Liposomes have been developed with improved serum stability and circulation half-times (see, for instance, U.S. Pat.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Quintanar-Guerrero et al., Drug Dev Ind Pharm 24(12):1113-1128, 1998; Quintanar-Guerrero et al., Pharm Res.
  • Injectable compositions can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No.5,466,468).
  • the form is sterile and fluid to the extent that it can be delivered by syringe.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • a coating such as lecithin
  • surfactants for example
  • the prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the preparation will include an isotonic agent(s), for example, sugar(s) or sodium chloride.
  • Prolonged absorption of the injectable compositions can be accomplished by including in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.
  • Injectable compositions can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. As indicated, under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
  • Sterile compositions can be prepared by incorporating the physiologically active component in an appropriate amount of a solvent with other optional ingredients (e.g., as enumerated above), followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized physiologically active components into a sterile vehicle that contains the basic dispersion medium and the required other ingredients (e.g., from those enumerated above).
  • compositions may be in liquid form, for example, as solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non- aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • Inhalable compositions can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions can 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. No. 5,770,219 and U.S. Pat. No. 5,783,208) and feedback-controlled delivery (U.S. Pat. No.5,697,899).
  • Supplementary active ingredients can also be incorporated into the compositions.
  • compositions can include at least 0.1% of the physiologically active components or more, although the percentage of the physiologically active components may, of course, be varied and may conveniently be between 1 or 2% and 70% or 80% or more or 0.5-99% of the weight or volume of the total composition.
  • the amount of physiologically active components in each physiologically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of compositions and dosages may be desirable.
  • compositions should meet sterility, pyrogenicity, and the general safety and purity standards as required by United States Food and Drug Administration (FDA) or other applicable regulatory agencies in other countries.
  • FDA United States Food and Drug Administration
  • the present disclosure includes cells including an artificial expression construct described herein.
  • a cell that has been transformed with an artificial expression construct can be used for many purposes, including in neuroanatomical studies, assessments of functioning and/or non-functioning proteins, and drug screens that assess the regulatory properties of enhancers.
  • a variety of host cell lines can be used, but in particular embodiments, the cell is a mammalian cell.
  • the artificial expression construct includes a regulatory element and/or a vector sequence of DLX2.0, minBglobin promoter, hSyn1 promoter, CMV promoter, hSyn1 promoter (shortened), 4x2C miR binding site, 8x2C miR binding site and/or eHGT_078h and/or CN3252, CN3254, CN3683, CN3684, CN3251, CN3253, CN3677, CN3678, CN4541, CN4542, CN4217, CN4218, CN4642, or CN4643, and the cell line is a human, primate, or murine cell.
  • Cell lines which can be utilized for transgenesis in the present disclosure also include primary cell lines derived from living tissue such as rat or mouse brains and organotypic cell cultures, including brain slices from animals such as rats, mice, non-human primates, or human neurosurgical tissue.
  • WO 91/13150 describes a variety of cell lines, including neuronal cell lines, and methods of producing them.
  • WO 97/39117 describes a neuronal cell line and methods of producing such cell lines.
  • the neuronal cell lines disclosed in these patent applications are applicable for use in the present disclosure.
  • neuronal describes something that is of, related to, or includes, neuronal cells. Neuronal cells are defined by the presence of an axon and dendrites.
  • neuronal-specific refers to something that is found, or an activity that occurs, in neuronal cells or cells derived from neuronal cells, but is not found in or occur in, or is not found substantially in or occur substantially in, non-neuronal cells or cells not derived from neuronal cells, for example glial cells such as astrocytes or oligodendrocytes.
  • non-neuronal cell lines may be used, including mouse embryonic stem cells. Cultured mouse embryonic stem cells can be used to analyze expression of genetic constructs using transient transfection with plasmid constructs. Mouse embryonic stem cells are pluripotent and undifferentiated.
  • LIF Leukemia Inhibitory Factor
  • Methods to differentiate stem cells into neuronal cells include replacing a stem cell culture media with a media including basic fibroblast growth factor (bFGF) heparin, an N2 supplement (e.g., transferrin, insulin, progesterone, putrescine, and selenite), laminin and polyornithine.
  • bFGF basic fibroblast growth factor
  • N2 supplement e.g., transferrin, insulin, progesterone, putrescine, and selenite
  • laminin e.g., transferrin, insulin, progesterone, putrescine, and selenite
  • laminin e.g., laminin and polyornithine.
  • U.S. Publication No.2012/0329714 describes use of prolactin to increase neural stem cell numbers while U.S. Publication No.2012/0308530 describes a culture surface with amino groups that promotes neuronal differentiation into neurons, astrocytes and oligodendrocytes.
  • BDNF brain derived growth factor
  • bFGF fibroblast growth factor
  • Patent No.6,103,530 somatostatin; amphiregulin; neurotrophins (e.g., cyclic adenosine monophosphate; epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); forskolin; GDNF family receptor ligands; potassium; retinoic acid (U.S. Patent No. 6,395,546); tetanus toxin; and transforming growth factor- ⁇ and TGF- ⁇ (U.S. Pat. Nos. 5,851,832 and 5,753,506).
  • neurotrophins e.g., cyclic adenosine monophosphate
  • GDNF family receptor ligands e.g., potassium
  • retinoic acid U.S. Patent No. 6,395,546
  • tetanus toxin transforming growth factor-
  • yeast one-hybrid systems may also be used to identify compounds that inhibit specific protein/DNA interactions, such as transcription factors for DLX2.0, minBglobin promoter, hSyn1, promoter, CMV promoter, hSyn1 promoter (shortened), 4x2C miR binding site, 8x2C miR binding site, and/or eHGT_078h.
  • Methods are also provided for administering a system of expression constructs to a subject in need thereof, which include administering a therapeutically effective amount of a system disclosed herein to a sample or subject comprising a targeted population of cells, and inducing expression of the first expression construct and the second expression construct of the system to reconstitute a sodium channel alpha subunit, thereby rescuing voltage-gated sodium channel function within the targeted population of cells.
  • the subject in need thereof has a sodium channelopathies, optionally comprising Dravet syndrome, myoclonic seizures, myoclonic astatic epilepsy (MAE), intractable childhood epilepsy with generalized tonic-clonic seizures, simple febrile seizures, generalized epilepsy and febrile seizures plus (GEFS+), migrating partial seizures of infancy, Lennox-Gastaut syndrome, or West syndrome.
  • the system of expression constructs is administered to a human subject or mammalian subject.
  • the system of expression constructs is administered to cells or tissue cultures obtained from, or derived from, a human subject or a mammalian subject.
  • Dravet syndrome is a severe early-onset epileptic encephalopathy marked by spontaneous and febrile seizures, motor disabilities, cognitive dysfunction, developmental delay, and heightened risk of premature death by sudden unexpected death in epilepsy (SUDEP).
  • DS afflicts approximately 1:16000 births, usually manifests in the first year of life, and produces profound symptoms that require life-long care.
  • Most first-line anti-epileptic drugs are ineffective or contraindicated for DS, although several recently approved drugs now partially ameliorate DS symptoms.
  • no FDA-approved long-term disease-modifying treatments currently exist for DS despite extensive efforts. As a result, a treatment for DS is a pressing unmet need for patients and their caregivers.
  • AAVs enhancer-adeno-associated viruses
  • Exemplary short intron sequences added are highlighted in italics or underlined in ‘hSCN1A-CO-Nterm1049- Intron,’ ‘hSCN1A-CO-Cterm949-Intron,’ ‘hSCN1A-CO-Nterm956-Intron,’ ‘hSCN1A-CO- Cterm1042-Intron,’ ‘hSCN1A-CO-Nterm947-Intron,’ and ‘hSCN1A-CO-Cterm1051-Intron’; or has a polynucleotide sequence of GTAAGTACTAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTCTATGGTTGGG ATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATA CCTCTTATCTTCCTCCCACAG (SEQ ID NO:107).
  • constructs were co-transfected with Na V 1.1 ⁇ -subunits (SCN1B/SCN2B).
  • SCN1A-FL full length SCN1A constructs show rapid and large inward sodium currents in response to depolarizing voltage steps
  • SCN1A-N+C SCN1A-N+C
  • mice After twenty days, we analyzed mouse motor cortex membrane protein content by western blot to assess efficiency of half joining by intein-mediated fusion.
  • a separate cohort of Dlx5/6-Cre; Ai14 mice to provide an independent label for telencephalic GABAergic interneurons we observed similar high levels of specificity and moderate completeness of AAV transduction (Fig. 9A-9C).
  • telencephalic GABAergic interneuron-specific SCN1A gene replacement could rescue DS symptoms in mouse models.
  • telencephalic GABAergic interneuron supplementation of AAV-mediated SCN1A transgene is sufficient to completely prevent SUDEP in DS model mice.
  • Dual DLX2.0 split-intein SCN1A vectors protect against thermal and spontaneous myoclonic and generalized tonic-clonic seizures in DS mice.
  • DS model mice are sensitive to thermally induced seizures, similar to patients with DS.
  • MC myoclonic
  • GTC generalized tonic-clonic
  • mice between P25 to P35 were analyzed DS mice between P25 to P35.
  • DLX2.0-SCN1A AAV vectors conferred strong and reproducible protection from mortality, induced seizures, and spontaneous seizure burden in two independent genetic mouse models of DS, although this protective effect of the AAV treatment is dose- and AAV quality- dependent.
  • Dual DLX2.0 vectors completely protect against SUDEP and seizures in mice with telencephalic GABAergic interneuron-specific Scn1a deletion.
  • Much more severe mortality has been seen with specific Scn1a loss in interneurons, likely due to a disrupted excitatory/inhibitory balance.
  • mice carrying the disease- causing mutation in the same telencephalic GABAergic interneuron population were generated by crossing mice carrying the Dlx5/6-Cre allele with mice carrying floxed Scn1a (Fig. 5A). Since the site of disease pathogenesis precisely matches the treatment target, in this experiment we directly tested the therapeutic effectiveness of the DLX2.0-SCN1A AAV vectors in a more precise rescue scenario.
  • telencephalic GABAergic interneuron-selective targeting is beneficial for gene replacement therapy in DS
  • a comparator we produced and tested split- intein vectors driven by hSyn1 which expresses in most brain neuronal populations, including excitatory and inhibitory neurons (Fig. 6A). Constructs for these vectors were built and packaged in the same way as DLX2.0 ones, except that hSyn1 promoter was used in this case.
  • GFAP astrogliosis
  • Iba1 microgliosis
  • VISp astrogliosis
  • mice two example mice spanning the range of astrogliosis observed are shown.
  • Nonselective neuronal expression of SCN1A exacerbated astrogliosis in DS model mice but did not cause overt changes in microglial appearance.
  • DLX2.0-SCN1A AAV vectors achieve long-term recovery of DS mortality (from 200 days to one year) in two independent DS mouse models at two independent testing sites. This robust mortality protection correlates with strong anti-seizure effect, indicating the mechanism behind mortality rescue is seizure reduction due to resupplying Na V 1.1 voltage- gated sodium channel activity.
  • the cell types sufficient to rescue DS epilepsy using SCN1A gene replacement [0218] Mouse models of DS indicate that epileptic symptomology is driven by a disruption of the excitatory/inhibitory balance, and congruently, patients display disrupted inhibition in the cortical microcircuit.
  • DS epilepsy is primarily a disease of the interneurons, and that interneuron targeting is an effective therapeutic strategy.
  • telencephalic GABAergic interneurons may contribute to the disease.
  • PVALB-expressing interneurons are thought to promote beneficial gamma rhythms, possibly through their Scn1a-dependent fast-spiking behavior.
  • SST-expressing and other telencephalic interneuron populations may also contribute to the epileptic phenotype.
  • DLX2.0 enhancer used in this study targets both PVALB and SST and also other telencephalic interneuron populations, in both mouse and human tissue, which may explain the strong anti-epileptic effect of DLX2.0-SCN1A.
  • Drug development to find pathway modulators that can overcome deficient Na V 1.1 has been challenging.
  • HC-AdVs also known as Helper-Dependent or “gutless”
  • our AAV-mediated and telencephalic GABAergic interneuron-selective gene replacement strategy is unique in several ways.
  • our vectors express a new copy of the SCN1A gene at moderate levels, and don’t upregulate the endogenous SCN1A allele, a strategy that could be ineffective or harmful for certain disease alleles.
  • the vectors target telencephalic GABAergic interneurons, the most essential cell type, whereas antisense oligonucleotides lack targeting ability. Since SCN1A is expressed in both excitatory and inhibitory neurons, activation of the SCN1A in excitatory neurons could attenuate the corrective effect of SCN1A upregulation.
  • telencephalic GABAergic interneurons Last, using AAV delivery, with the PHP.eB capsid and neonatal ICV injection, widespread viral transduction of telencephalic GABAergic interneurons is achieved. We are not aware of a vector that can give superior widespread transduction to the brain. None of the previous studies delivering or activating Na V 1.1 using exogenous agents have demonstrated complete recovery of mortality as we have observed. Moreover, we have demonstrated robust rescue in two genetic models, at two research sites, and even in a severe Dlx5/6-Cre-driven knockout model. Thus, gene replacement, cell type specificity, and broad coverage of telencephalic interneurons provides a unique and highly effective treatment for DS.
  • the split-intein fused SCN1A halves are delivered in advanced BBB-penetrant AAV capsids.
  • the AAV capsids comprise AV-PHP.eB, which efficiently transduce the central nervous systems.
  • the AAV capsids comprise AAV-PHP.S, which efficiently transduce the peripheral nervous systems.
  • the split-intein fused SCN1A halves delivered in AAV capsids are administered locally with intraparenchymal delivery.
  • regulatory elements that confer a broader distribution pattern extending to sub-telencephalic regions may be used to further treat non-epileptic DS.
  • regulatory elements known capable of restricting expression to interneurons include the distal-less homeobox 5 and 6 (Dlx5/6) genes, which are specifically expressed by all forebrain GABAergic interneurons during embryonic development. These genes have an inverted orientation relative to one another and share a 400bp (mI56i or mDlx) and a 300bp (mI56ii) enhancer sequence in the 10kb non-coding intergenic region 3′ to each of them. The high degree of conservation of these sequences across vertebrate species is indicative of an important role in gene regulation.
  • the mDlx enhancer can be used to target reporter genes in a pattern very similar to the normal patterns of Dlx5/6 expression during embryonic development, e.g., selectively expressed within GABAergic interneurons in a wide variety of vertebrate species.
  • Materials and Techniques [0225] Study design.
  • mice at SCRI were maintained in standard cages for laboratory mice, on a 12h:12h light-dark cycle, with ad-libitum access to food and water, at 23 degrees C.
  • Mouse models and their littermates of DS used in these studies were generated using Cre-Lox technology. DS mice carrying a whole body heterozygous knock-out of Scn1a were obtained by breeding floxed Scn1a mice with Meox2-Cre mice (Strain #: 003755; Jackson Laboratories).
  • mice carrying an Scn1a KO allele restricted to (specifically in) forebrain GABAergic neurons alone were generated by breeding floxed Scn1a mice with Dlx5/6-Cre mice (Strain#: 008199, Jackson Laboratories). All breeder mice were maintained on a C57BL/6J background for at least 10 generations.
  • Neonatal P0-3 mice were cryo-anesthetized on a small aluminum plate placed on ice.
  • Single AAVs or dual AAVs were injected bilaterally into lateral ventricles using a 33-gauge needle attached to Hamilton microliter syringe.
  • 2.5 ⁇ l of the AAV solution were injected in each ventricle for a total of 5 ⁇ l per mouse containing a total of 1e10 or 3e10 gc each viral vector.
  • mice were put back into their nest and placed on a warming pad until their body temperature returned to normal.
  • Electrocorticography (ECoG) electrode implantation surgery [0235] Mice underwent survival surgery to implant ECoG and EMG electrodes, under isoflurane anesthesia. A midline incision was made above the skull to expose the site of electrode implantation.
  • ECoG electrodes consisted of a micro-screw attached to a silver wire (diameter: 130 ⁇ m bare; 180 ⁇ m coated). EMG electrodes were made of a silver wire shaped in a loop at one end. An ECoG electrode micro-screw was inserted into a small cranial burr hole above the somatosensory cortex in each hemisphere.
  • a reference electrode micro- screw was placed in a burr hole above the cerebellum. EMG electrodes were inserted and secured into the neck muscles. All electrodes were attached to an interface connector and the assembly was affixed to the skull with dental cement (Lang Dental Manufacturing Co., Inc., Wheeling, IL, United States). The incision around the electrode implant was closed using sutures. Mice were allowed to recover from surgery for 1–3 days before recording. [0236] Video-ECoG-EMG recording. [0237] Simultaneous video-ECoG-EMG records were collected in conscious mice on a PowerLab 8/35 data acquisition unit using LabChart 8.0 software (AD Instruments, Colorado Spring, Co). All bioelectrical signals were acquired at 1-KHz sampling rate.
  • the ECoG signals were processed with a 1–70 Hz bandpass filter and the EMG signals with a 10-Hz highpass filter. Power-spectral densities of the electrical signals were computed, and video-ECoG-EMG records were inspected for interictal spikes and ictal epileptiform events. Interictal spikes were characterized on ECoG as discharges with an abrupt onset, a sharp contour, and an amplitude greater than twice the background activity. Interictal spikes were frequently followed by a slow wave, but they were not associated with increased EMG activity or movement on video. Conversely, GTC seizure events were marked at their onset by bursts of generalized spikes and waves of increasing amplitude and decreasing frequency on ECoG.
  • mice were handled under appropriate institutional protocols and guidelines. Procedures were approved by the Allen Institute Institutional Animal Care and Use Committee under protocols 2002 and 2301. We housed animals in a 14:10 light:dark cycle in ventilated racks with ad libitum access to food (LabDiet 5001) and water, as well as enrichment items consisting of plastic shelters and nesting materials. Young animals are weaned promptly at 21 days of age.
  • mice from Jackson Laboratory (strain # 034129) and maintained breeders on a 129S1/SvImJ genetic background.
  • C57Bl/6J mice Jackson strain # 000664
  • Genotyping was performed with tail biopsy at P2, and we utilized PCR-sanger genotyping services at Transnetyx for this line.
  • Neonatal ICV injection we used the neonatal intracerebroventricular (ICV) injection technique. Briefly, we anesthetized P2 neonates with ice but shielded from direct ice exposure. During anesthesia, pups were injected freehand bilaterally with 5 ⁇ L (2.5 ⁇ L each hemisphere) of AAV-containing solution using a Hamilton syringe. AAVs were diluted in sterile PBS to expel either 1e10 or 3e10 gc of each of two halves of the dual-vector encoding split-intein human SCN1A.
  • ICV neonatal intracerebroventricular
  • mice In control animals, only one half of the dual-vector system was delivered, or DLX2.0-SYFP2-only empty control vector (CN1390), or mice were left untreated. Control vectors were delivered at 3e10 gc per animal with BL-ICV delivery. After injection, pups were gently warmed on a cage warmer set to 28°C with mother present. [0241] Continuous video ECoG/EMG recordings. [0242] We implanted adult mice (P56-90) with ECoG/EMG headmount. For stereotaxic surgical procedures, we induced anesthesia in mice first with 5% isoflurane in oxygen, and then maintained anesthesia with 1.5-2.5% isoflurane.
  • Electrode leads were soldered onto the 8-pin headmount (#8431-SM, Pinnacle Technology Inc.).
  • the headmount contains two insulated EMG wire electrodes that are pre-soldered, and these EMG electrodes were inserted into the neck muscles. All wires, pins and the headmount were embedded in light curable dental composite resin (Prime-Dent, Prime Dental Manufacturing Inc., Chicago, IL, USA). Mice were singly housed post-surgery and recovered for at least 7 days prior to recording. Recordings were thus acquired between ages P74 and P123. For recordings at AIBS, mice were singly housed in 10-inch clear acrylic chambers (#8228, Pinnacle Technology Inc.) under a 14-hr on, 10-hr off light/dark cycle.
  • mice were tethered with the pre-amplifier through a commutator to the data acquisition system (#8401-HR, Pinnacle Technology Inc.). All ECoG/EMG data were recorded with a 500 Hz sampling rate, 10 X gain, a low pass (ECoG: 0.5 Hz; EMG: 1 Hz) filter, and a high pass (500 Hz) filter. Videos were recorded synchronously at a frame rate of 10 frame/s with a resolution of 640x480 pixels. We implanted a total of 18 non-injected Scn1a +/R613X mice. Of these 18, seven mice (3M+4F) died during recovery prior to recording, and we recorded from the remaining 11 mice (9M+2F).
  • mice monoclonal anti-FLAG clone M2 (Millipore-Sigma # F1804)
  • rabbit monoclonal anti-HA clone C29F4 (1/1000, Cell Signaling # 3724S)
  • mouse monoclonal anti-HA clone 16B12 (1/1000, Biolegend # 901513)
  • mouse monoclonal anti-HA clone HA.C5 (1/1000, Thermo Fisher Scientific # MA5-27543
  • mouse monoclonal anti-Gad67 clone 1G10.2 (1/250, Millipore-Sigma # MAB5406)
  • mouse monoclonal anti-NeuN clone 1B7 (1/500, Novus Biologicals # NBP1-92693AF647)
  • guinea pig polyclonal anti-GABA (1/500, Millipore-
  • This residue is alanine in the NCBI RefSeq sequence but is a threonine in commercial clones available from Origene (catalog # RG220167), as well as a conserved threonine across most other mammalian species (Fig.8C), and finally we observed this residue to be a threonine in three of three human tissue donors sequenced in our prior work (Fig. 8D, data available at dbGaP # phs002292.v1.p1). From the Genome Aggregation Database (gnomAD) the reference alanine allele appears to be the minor allele in the human population (27%), whereas the majority of alleles in the population encode threonine at that position (73%).
  • gnomAD Genome Aggregation Database
  • ORF full protein open reading frame
  • the full length human SCN1A pSMART vector contained only a C-terminal FLAG epitope but not an N-terminal HA epitope.
  • pAAV vectors For cloning into pAAV vectors we inserted the intein-fusion halves into CN1390 (Addgene plasmid #163505) in place of SYFP2 reporter for DLX2.0-driven expression or into CN1839 (Addgene plasmid #163509) in place of SYFP2- 10aa-H2B for hSyn1-driven expression. During this cloning we also replaced BGH polyA in the original vectors sequences with shorter synthetic polyA sequences due to size constraints.
  • plasmids containing the split fusion protein halves alone did not exhibit rearrangements or require special culturing techniques, and we amplified these plasmids in either pSMART-HC-Kan or pAAV backbone using Stbl3 cells (Thermo Fisher Scientific # C737303).
  • HEK293 cells (CRL-1573; ATCC, Gaithersburg, MD) were cultured in standard media consisting of DMEM, high glucose, glutaMAX (Gibco 10566016; Thermo Fisher Scientific, Waltham, MA), supplemented with 10% (v/v) fetal calf serum (FCS) (Gibco A5670401; Thermo Fisher Scientific, Waltham, MA) and 1% (v/v) Penicillin-Streptomycin (P/S) (10,000 U/ml) (Gibco 15140148; Thermo Fisher Scientific, Waltham, MA), grown at 37°C and 5% CO 2 .
  • FCS fetal calf serum
  • P/S Penicillin-Streptomycin
  • Plasmid constructs were acutely transfected into HEK293 cells using Viafect reagent (E4981; Promega, Madison, WI), following the manufacture’s protocol.
  • HEK293 cells were first prepared for transfection by plating into 12-well tissue culture plates (Nunc 12-565- 321; Thermo Fisher Scientific, Waltham, MA) at a density of ⁇ 0.5-2 x10 5 cells per well and grown to ⁇ 80-90% confluence with standard media, allowing for one confluent well per transfection condition. On the day of transfection, media in confluent wells to be transfected were replaced with 0.5 mL fresh DMEM with 10% FCS, without P/S. Lipophilic/DNA transfection complexes were generated for each well to be transfected.
  • plasmid DNAs used for transfections per well: a) Full- length SCN1A (SCN1A-FL) 0.8 ⁇ g of pDNA, b) SCN1A-Ntm (SCN1A-N) 0.4 ⁇ g pDNA combined with SCN1A-Ctm (SCN1A-C) 0.4 ⁇ g pDNA, c) SCN1A-Ntm (SCN1A-N) 0.8 ⁇ g pDNA, d) SCN1A-Ctm (SCN1A-C) 0.8 ⁇ g pDNA, e) empty vector (SYFP only) 0.8 ⁇ g pDNA.
  • transfection conditions were performed in a background of 80 ng pDNA of a bi- cistronic construct expressing SCN1B and SCN2B ( ⁇ 1/2), which encodes two sodium channel ⁇ -subunits, yielding an ⁇ : ⁇ subunit-encoding pDNA mass ratio of 10:1.
  • One additional control utilized only 80 ng of ⁇ 1/2 pDNA.
  • composition of recording solutions was: Bath (in mM, 140 NaCl, 2 CaCl2, 2 MgCl2, 10 HEPES, pH 7.4); Pipette internal solution (in mM, 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES, pH 7.4). Patch pipettes were pulled from borosilicate glass (1B120F-4; World Precision Instruments, Sarasota, FL) on a P-97 Sutter Instruments puller (Novato, CA), and fire-polished on a Micro-Forge MF-830 (Narashige International USA, Amityville, NY) to a resistance of 0.8-1.5 M ⁇ .
  • G conductance/voltage (G/V) plots
  • I peak current
  • V m membrane potential
  • E Na-re v Na + reversal potential.
  • Na + reversal potential was 35 mV, based on a calculated Nernst equilibrium potential with the recording solutions used.
  • Peak currents were recorded in response to a family of voltage steps from a holding potential of -120 mV to 40 mV, in 5 mV increments, with an inter-pulse interval of 2 seconds to allow channels to fully deactivate to the deep closed state.
  • the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

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Abstract

L'invention concerne des systèmes et des procédés pour l'administration et l'expression médiées par AAV à médiation par intéine divisée de grandes molécules de protéine présentant des caractéristiques de sécurité améliorées. Spécifiquement, des séquences de dégron en tant que signaux de dégradation sont incluses à une extrémité N ou C spécifique de fragments d'intéine divisée qui sont chacun couplés à une partie de la séquence de codage de sous-unité alpha de canal sodique sensible à la tension (par exemple, sous-unité alpha 1, Nav1.1), et lors de l'expression et de l'épissage de fragments d'intéine divisée, Nav1.1 est reconstitué tandis que l'intéine trans-épissée est digérée conjointement avec le dégron couplé par l'intermédiaire de voies de dégradation de dégron.
PCT/US2024/042744 2023-08-16 2024-08-16 Systèmes et procédés pour améliorer la sécurité d'une thérapie génique médiée par aav à intéine divisée Pending WO2025038955A1 (fr)

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